Heteroatom containing deoxyuridine triphosphatase inhibitors

ABSTRACT

Provided herein are dUTPase inhibitors, compositions comprising such compounds and methods of using such compounds and compositions in a method of treating cancer. The dUTPase inhibitors disclosed contain a uracil isostere in the molecule represented by a 2,6-diketopiperazine moiety. Thioanalogs of the uracil isostere where a thione replaces each of the ketone are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/109,616, filed Jul. 1, 2016, now U.S. Pat. No. 9,790,214, which is aNational Stage Entry of International Patent Application No.PCT/US2015/010059, filed Jan. 2, 2015, which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 61/923,534,filed Jan. 3, 2014, the contents of each of which are incorporatedherein by reference in their entirety.

BACKGROUND

Thymidylate metabolism is required for producing essential buildingblocks necessary to replicate DNA in dividing cells and has long been animportant therapeutic target for cornerstone cancer drugs. Drugstargeting this pathway such as 5-fluorouracil (5-FU) inhibit the enzymethymidylate synthase (TS) and are currently critical standard-of caretherapies. TS-targeted agents are used for the treatment of a variety ofcancers including colon, gastric, head and neck, breast, lung and bloodrelated malignancies among others. Grem, J. L., 5-Fluorouracil plusleucovorin in cancer therapy, in Principals and Practice of OncologyUpdate Series, J. De Vita, V. T., S. Hellman, and A. Rosenberg, Editors.1988, J. B. Lippincott: Philadelphia, Pa.

There are two classes of drugs that target the TS enzyme: thefluoropyrimidines and the antifolates. The fluoropyrimidines, 5-FU, S-1and capecitabine (Xeloda®), have wide use in the treatment ofgastrointestinal and breast cancers, while the antifolate pemetrexed(Alimta®) is currently used for the treatment of non-small cell lungcancer (NSCLC). Since the discovery of 5-FU over fifty years ago byCharles Heidelberger, the fluoropyrimidines remain one of the mostcommon and effective anticancer cancer drugs used worldwide. Due to thisfact, there is an abundance of clinical experience and insight into themechanism of action of these agents.

The TS inhibitor 5-fluorouracil (5 FU) remains the foundation of manyfirst and second line regimens in the treatment of colon cancer. Singleagent therapies including oxaliplatin, irinotecan, Erbitux and Avastin,demonstrate lowered activity in colon cancer compared to 5-FU. Inaddition to colon cancer, TS-inhibitory agents have demonstratedefficacy in several other solid tumor types. Standard of care nowincorporates 5-FU as the backbone drug in combination with oxaliplatinor irinotecan or another agent.

Deoxyuridine triphosphatase (“dUTPase”) is a ubiquitous enzyme that isessential for viability in both prokaryotic and eukaryotic organisms; asthe main regulator of dUTP pools, the expression of dUTPase could haveprofound effects on the utility of chemotherapeutics that inhibitthymidylate biosynthesis. Normally, dUTPase mediates a protective roleby limiting the expansion of dUTP pools and countering the cytotoxiceffect of uracil misincorporation. According to this model, elevatedlevels of dUTPase could prevent TS inhibitor-induced dUTP accumulationand induce drug resistance. It has been shown that dUTPase overexpression results in a significant decrease in dUTP accumulation andincreased resistance to drug treatment when compared to controls.

Chemotherapeutic agents that target de novo thymidylate metabolism arecritical for the treatment of a variety of solid tumors, howeverclinical efficacy is often hindered by drug resistance. Becauseresistance to these agents is a common occurrence, the identificationand exploitation of novel determinants of drug sensitivity within thispathway of proven therapeutic utility is important. As disclosed byLadner et al. in U.S. Patent Publ. No. US 2011/0212467, the dUTPaseenzyme and the uracil-DNA misincorporation pathway can play a drivingrole in mediating cytotoxicity to TS-directed chemotherapies.

For example, nearly half of cancer patients do not benefit from5-FU-based treatment due to intrinsic or acquired drug resistance. Dueto this fact, there is a critical need to overcome the fundamentalchallenge of drug resistance and provide new therapeutic strategies toimprove patient outcome. This disclosure satisfies this need andprovides related advantages as well.

SUMMARY

In some aspects, this disclosure provides compounds, compositions andmethods that inhibit dUTPase when used alone or in combination with atleast one dUTPase-directed chemotherapy. In some aspects, thisdisclosure provides compounds, compositions and methods for treatingcancer, killing cancer cells, and inhibiting cancer cell growth whenused in combination with at least one TS-directed chemotherapy.Compounds of this class include the following compounds of formulas (I),(II), and (III).

Thus, in one aspect, provided herein are compounds of formulas (I) and(II):

-   or a tautomer thereof, or a pharmaceutically acceptable salt and/or    a solvate thereof, or a stereochemically pure or enriched    stereoisomer of each thereof, wherein

-   is a uracil isostere-   which is a 6 membered heterocycle comprising a —C(═V)—NH—C(═V)—    moiety wherein V is independently O or S, and at least another ring    heteroatom, and wherein the heterocyclic ring is optionally    substituted, as provided hereinbelow;-   W is a bond or optionally substituted —CH₂—;-   X is a bond, O, S, NR¹⁹, optionally substituted C₁-C₆ alkylene,    optionally substituted C₂-C₆ alkenylene, or optionally substituted    C₂-C₆ alkynylene group, a divalent optionally substituted C₆-C₁₀    aromatic hydrocarbon group, or a divalent optionally substituted    saturated or unsaturated C₂-C₁₀ heterocyclic or optionally    substituted C₁-C₁₀ heteroaryl group;-   R¹⁹ is hydrogen, optionally substituted C₁-C₆ alkyl or optionally    substituted C₃-C₈ cycloalkyl;-   Y is a bond or an optionally substituted C₁-C₁₀ alkylene which    further optionally has a cycloalkylidene structure on one carbon    atom, or is optionally substituted C₂-C₆ alkenylene, or optionally    substituted C₂-C₆ alkynylene group, or Y is -L¹⁰-B¹-L¹¹-;-   L¹⁰ and L¹¹ independently are optionally substituted C₁-C₆ alkylene,    optionally substituted C₂-C₆ alkenylene, or optionally substituted    C₂-C₆ alkynylene group;-   B¹ is a divalent optionally substituted C₆-C₁₀ aromatic hydrocarbon    group, or a divalent optionally substituted saturated or unsaturated    C₂-C₁₀ heterocyclic or optionally substituted C₁-C₁₀ heteroaryl    group;-   Z is —PO₂—NR³¹R³², —SO₂NR³¹R³², —NR³PO₂—R⁴, —NR³SO₂—R⁴, or R⁴    wherein R³¹ and R³² are the same or different and each represents a    hydrogen atom, optionally substituted C₁-C₆ alkyl group optionally    substituted with an aryl group, wherein the aryl group, together    with the R³¹ or R³², may form a condensed bicyclic hydrocarbon, or    R³¹ and R³² are taken together with the adjacent nitrogen atom form    an optionally substituted C₂-C₁₀ heterocyclic group or an optionally    substituted C₁-C₁₀ heteroaryl group;-   Z¹ is —PO₂—NR³¹R³² or —(OR³)P(O)—R⁴ wherein R³¹ and R³² are    independently a hydrogen atom, optionally substituted C₁-C₆ alkyl    group optionally substituted with an aryl group, wherein the aryl    group, together with the R³¹ or R³², may form a condensed bicyclic    hydrocarbon, or R³¹ and R³² taken together with the adjacent    nitrogen atom form an optionally substituted C₂-C₁₀ heterocyclic    group or an optionally substituted C₁-C₁₀ heteroaryl group;-   R³ is hydrogen or optionally substituted C₁-C₆ alkyl; and-   R⁴ is optionally substituted C₆-C₁₀ aryl, an optionally substituted    C₂-C₁₀ heterocyclic group, or an optionally substituted C₁-C₁₀    heteroaryl group.

In another aspect, provided herein are compounds of formula (III):

-   wherein A is

-   each V is independently O or S,-   R¹¹ is hydrogen, halo, R¹² or —O—R¹², wherein R¹² is C₁-C₆ alkyl,    C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted with 1-3    hydroxy, fluoro, chloro, and amino substituent,-   r is 1, 2, or 3,-   Y¹⁰ is O, S, SO, SO₂, NH or NR¹⁵, or L¹ is joined at Y¹⁰ with Y¹⁰    being a nitrogen atom;-   R¹⁵ is C₁-C₆ alkyl optionally substituted with 1-3 C₁-C₆ alkoxy,    hydroxy, amino, and oxo groups,-   L¹- is

-   Y¹ is CH₂, O, S,-   X¹⁰ is NH, NCO₂R²⁰, O, —CO—, —CO—NH—, or CH₂,-   R²⁰ is C₁-C₆ alkyl optionally substituted with 1-3 C₆-C₁₀ aryl    groups,-   u is 0, 1, 2, 3, or 4,-   R^(z) is hydroxy or hydrogen,-   R^(w) is C₁-C₆ alkyl or hydrogen, and-   the phenylene and the heteroarylene rings are optionally    substituted,-   Z is phenyl or a 5 or 6 member heteroaryl substituted with an R⁶ and    an R⁶⁰ groups, wherein the R⁶ and the R⁶⁰ are positioned 1,2 with    respect to each other,-   R⁶ is hydrogen, optionally substituted C₁-C₆ alkoxy, or halo, and-   R⁶⁰ is —OR⁷ or —NHR⁷R⁷⁰,-   R⁷ is optionally substituted C₁-C₁₀ alkyl, optionally substituted    C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl, optionally    substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₁₀    heteroaryl, optionally substituted C₃-C₁₀ heterocyclyl, or    optionally substituted phenyl, and-   R⁷⁰ is hydrogen or R⁷.

This disclosure also provides a tautotomer, or its pharmaceuticallyacceptable salt and/or a solvate of a compound as disclosed herein.Methods to prepare such are known in the art.

This disclosure also provides a stereochemically pure enantiomer of acompound as described herein, its tautotomer, diastereoisomer or itspharmaceutically acceptable salt and/or a solvate thereof. Methods topurify and identify the pure enantiomer are known in the art anddescribed herein.

In another aspect, compositions comprising one or more compounds of theabove-noted provided and a carrier are provided herein. In oneembodiment, the composition is a pharmaceutical composition andtherefore further comprise at least a pharmaceutically acceptablecarrier or a pharmaceutically acceptable excipicnt. The compositions areformulated for various delivery modes, e.g., systemic (oral) or local.

In another aspect, this disclosure provides compositions comprising oneor more compounds as provided herein and a dUTPase-directed chemotherapyand a carrier, such as a pharmaceutically acceptable carrier. Thecompound and chemotherapy can be in varying amounts, and in one aspect,each in an effective amount when used in combination, provides atherapeutic benefit as described herein. The compositions are formulatedfor various delivery modes, e.g., systemic (oral) or local.

In another aspect, methods are provided for inhibiting deoxyuridinetriphosphatase (dUTPase) comprising contacting the dUTPase with aneffective amount of a compound or a composition provided herein. Inanother aspect, the method further comprises contacting the dUTPase witha dUTPase-directed chemotherapy alone or in combination with thecompound as provided herein. The contacting can be in vitro, in vivo,simultaneous or concurrent. In a further aspect the dUTPase-directedchemotherapy is contacted prior to the compound or composition asdescribed herein. In another aspect, the dUTPase-directed chemotherapyis contacted subsequent to the compound or composition. In a yet furtheraspect, the compound or composition and the dUTPase-directedchemotherapy are sequentially administered through several rounds oftherapy. The contacting can be simultaneous or concurrent and/or invitro (cell free), ex vivo or in vivo. In a further aspect, thecompounds or compositions of this disclosure are administered to apatient identified or selected for the therapy by determing that thepatient has a tumor or mass that over expresses dUTPase. Methods toidentify such patients are known in the art and incorporated herein. Themethods when administered to a subject such as a human patient, can befirst line, second line, third line, fourth line or further therapy.

Also provided is a method for reversing resistance to a dUTPase-directedchemotherapy comprising contacting a cell overexpressing dUTPase with aneffective amount of a compound or a composition provided herein, aloneor in combination with a dUTPase-directed chemotherapy. In one aspect,the cell is first identified as overexpressing dUTPase by a screen asdisclosed by U.S. Pat. No. 5,962,246. In another aspect, the methodfurther comprises subsequently contacting the cell expressing dUTPasewith a dUTPase-directed chemotherapy. The methods can be administered assecond line, third line, fourth line or further therapy.

Further provided is a method for enhancing the efficacy of adUTPase-directed chemotherapy comprising contacting a cell, e.g., in oneaspect a cell over expressing dUTPase, with an effective amount of acompound or a composition provided herein. In another aspect, the methodfurther comprises contacting the cell with a dUTPase-directedchemotherapy. The contacting can be simultaneous or concurrent and/or invitro (cell free), ex vivo or in vivo. In a further aspect, thedUTPase-directed chemotherapy is contacted prior to the compound orcomposition as described herein, or vice versa. The methods whenadministered to a subject such as a human patient, can be first line,second line, third line, fourth line or further therapy.

In another aspect, provided herein is a method of treating a diseaseassociated with the dUTPase pathway, e.g., cancer, viral infection,bacterial infection, or an autoimmune disorder, comprising administeringto a patient in need of such treatment an effective amount of thecompound provided herein or a composition provided herein in combinationwith an agent which is suitable for treating the disease, therebytreating the disease. The administration of the compound of thisinvention and the agent that is suitable for the disease (e.g., adUTPase inhibitor) can be simultaneous or concurrent and/or in vitro(cell free), ex vivo or in vivo. In a further aspect the agent that issuitable for treating the disease is administered prior to the compoundor composition as described herein, or vice versa. In one aspect, thepatient being treated is selected for the therapy by screening a cell ortissue sample isolated from the patient for over expression of dUTPase.The therapy is then administered to this patient after the screen, andthe patient has been selected for therapy.

In another aspect, provided herein is a kit comprising a compoundprovided herein or a composition provided herein. The kit can furthercomprise one more of a dUTPase inhibitor (e.g., an antitumor agent) andinstructions for administering the agent. Yet further provided in thekit are reagents and instructions to screen for dUTPase expression.

In each of the above embodiments, a non-limiting example of the dUTPasemediated chemotherapy comprises a TS-inhibitor, e.g., 5-FU or 5-FUcontaining therapy such as 5-FU based adjuvant therapy and chemicalequivalents thereof.

DETAILED DESCRIPTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure in their entirety to more fully describe the state of the artto which this invention pertains.

Definitions

The practice of the present technology will employ, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,immunology, molecular biology, microbiology, cell biology andrecombinant DNA, which are within the skill of the art. See, e.g.,Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual,2^(nd) edition (1989); Current Protocols In Molecular Biology (F. M.Ausubel, et al. eds., (1987)); the series Methods in Enzymology(Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson,B. D. Hames and G. R. Taylor eds. (1995)), Harlow and Lane, eds. (1988)Antibodies, a Laboratory Manual, and Animal Cell Culture (R. I.Freshney, ed. (1987)).

As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompounds, compositions and methods include the recited elements, butnot exclude others. “Consisting essentially of” when used to definecompounds, compositions and methods, shall mean excluding other elementsof any essential significance to the combination. Thus, a compositionconsisting essentially of the elements as defined herein would notexclude trace contaminants, e.g., from the isolation and purificationmethod and pharmaceutically acceptable carriers, preservatives, and thelike. “Consisting of” shall mean excluding more than trace elements ofother ingredients. Embodiments defined by each of these transition termsare within the scope of this technology.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1, 5, or 10%. It is to be understood,although not always explicitly stated that all numerical designationsare preceded by the term “about.” It also is to be understood, althoughnot always explicitly stated, that the reagents described herein aremerely exemplary and that equivalents of such are known in the art.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms.This term includes, by way of example, linear and branched hydrocarbylgroups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—),isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—),sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—), n-pentyl(CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

“Alkenyl” refers to monovalent straight or branched hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of vinyl (>C═C<)unsaturation. Such groups are exemplified, for example, by vinyl, allyl,and but-3-en-1-yl. Included within this term are the cis and transisomers or mixtures of these isomers.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of acetylenic (—C≡C—)unsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

“Substituted alkyl” refers to an alkyl group having from 1 to 5,preferably 1 to 3, or more preferably 1 to 2 substituents selected fromthe group consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy,substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substitutedcycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substitutedheteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio,substituted heteroarylthio, heterocyclic, substituted heterocyclic,heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,substituted heterocyclylthio, nitro, SO₃H, substituted sulfonyl,substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substitutedalkylthio, wherein said substituents are as defined herein.

“Substituted alkenyl” refers to alkenyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy,substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substitutedcycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,guanidino, substituted guanidino, halo, hydroxyl, heteroaryl,substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy,heteroarylthio, substituted heteroarylthio, heterocyclic, substitutedheterocyclic, heterocyclyloxy, substituted heterocyclyloxy,heterocyclylthio, substituted heterocyclylthio, nitro, SO₃H, substitutedsulfonyl, substituted sulfonyloxy, thioacyl, thiol, alkylthio, andsubstituted alkylthio, wherein said substituents are as defined hereinand with the proviso that any hydroxyl or thiol substitution is notattached to a vinyl (unsaturated) carbon atom.

“Substituted alkynyl” refers to alkynyl groups having from 1 to 3substituents, and preferably 1 to 2 substituents, selected from thegroup consisting of alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy,aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,substituted aryl, aryloxy, substituted aryloxy, arylthio, substitutedarylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxylester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy,substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio,cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substitutedcycloalkenyloxy, cycloalkenylthio, substituted cycloalkenylthio,guanidino, substituted guanidino, halo, hydroxy, heteroaryl, substitutedheteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio,substituted heteroarylthio, heterocyclic, substituted heterocyclic,heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio,substituted heterocyclylthio, nitro, SO₃H, substituted sulfonyl,substituted sulfonyloxy, thioacyl, thiol, alkylthio, and substitutedalkylthio, wherein said substituents are as defined herein and with theproviso that any hydroxyl or thiol substitution is not attached to anacetylenic carbon atom.

“Alkylene” refers to divalent saturated aliphatic hydrocarbyl groupspreferably having from 1 to 6 and more preferably 1 to 3 carbon atomsthat are either straight-chained or branched. This term is exemplifiedby groups such as methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)— or —CH(CH₃)CH₂—), butylene(—CH₂CH₂CH₂CH₂—), isobutylene (—CH₂CH(CH₃)CH₂—), sec-butylene(—CH₂CH₂(CH₃)CH—), and the like. Similarly, “alkenylene” and“alkynylene” refer to an alkylene moiety containing respective 1 or 2carbon carbon double bonds or a carbon carbon triple bond.

“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents selected from the group consistingof alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl,substituted aryl, aryloxy, substituted aryloxy, cyano, halogen,hydroxyl, nitro, carboxyl, carboxyl ester, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic,substituted heterocyclic, and oxo wherein said substituents are definedherein. In some embodiments, the alkylene has 1 to 2 of theaforementioned groups, or having from 1-3 carbon atoms replaced with—O—, —S—, or —NR^(Q)— moieties where R^(Q) is H or C₁-C₆ alkyl. It is tobe noted that when the alkylene is substituted by an oxo group, 2hydrogens attached to the same carbon of the alkylene group are replacedby “═O”. “Substituted alkenylene” and “substituted alkynylene” refer toalkenylene and substituted alkynylene moieties substituted withsubstituents as described for substituted alkylene.

“Alkoxy” refers to the group —O-alkyl wherein alkyl is defined herein.Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, and n-pentoxy.

“Substituted alkoxy” refers to the group —O-(substituted alkyl) whereinsubstituted alkyl is defined herein.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclic-C(O)—, and substitutedheterocyclic-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted awl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein. Acyl includes the“acetyl” group CH₃C(O)—.

“Acylamino” refers to the groups —NR⁴⁷C(O)alkyl, —NR⁴⁷C(O) substitutedalkyl, —NR⁴⁷C(O)cycloalkyl, —NR⁴⁷C(O) substituted cycloalkyl,—NR⁴⁷C(O)cycloalkenyl, —NR⁴⁷C(O) substituted cycloalkenyl,—NR⁴⁷C(O)alkenyl, —NR⁴⁷C(O) substituted alkenyl, —NR⁴⁷C(O)alkynyl,—NR⁴⁷C(O) substituted alkynyl, —NR⁴⁷C(O)aryl, —NR⁴⁷C(O) substitutedaryl, —NR⁴⁷C(O)heteroaryl, —NR⁴⁷C(O) substituted heteroaryl,—NR⁴⁷C(O)heterocyclic, and —NR⁴⁷C(O) substituted heterocyclic whereinR⁴⁷ is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Acyloxy” refers to the groups alkyl-C(O)O—, substituted alkyl-C(O)O—,alkenyl-C(O)O—, substituted alkenyl-C(O)O—, alkynyl-C(O)O—, substitutedalkynyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—,substituted cycloalkyl-C(O)O—, cycloalkenyl-C(O)O—, substitutedcycloalkenyl-C(O)O—, heteroaryl-C(O)O—, substituted heteroaryl-C(O)O—,heterocyclic-C(O)O—, and substituted heterocyclic-C(O)O— wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

An animal, subject or patient for diagnosis or treatment refers to ananimal such as a mammal, or a human, ovine, bovine, feline, canine,equine, simian, etc. Non-human animals subject to diagnosis or treatmentinclude, for example, simians, murine, such as, rat, mice, canine,leporid, livestock, sport animals, and pets.

“Amino” refers to the group —NH₂.

“Substituted amino” refers to the group —NR⁴⁸R⁴⁹ where R⁴⁸ and R⁴⁹ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl,—SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-cycloalkenyl,—SO₂-substituted cylcoalkenyl, —SO₂-aryl, —SO₂-substituted aryl,—SO₂-heteroaryl, —SO₂-substituted heteroaryl, —SO₂-heterocyclic, and—SO₂-substituted heterocyclic and wherein R⁴⁸ and R⁴⁹ are optionallyjoined, together with the nitrogen bound thereto to form a heterocyclicor substituted heterocyclic group, provided that R⁴⁸ and R⁴⁹ are bothnot hydrogen, and wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein. When R⁴⁸ is hydrogen and R⁴⁹ isalkyl, the substituted amino group is sometimes referred to herein asalkylamino. When R⁴⁸ and R⁴⁹ are alkyl, the substituted amino group issometimes referred to herein as dialkylamino. When referring to amonosubstituted amino, it is meant that either R⁴⁸ or R⁴⁹ is hydrogenbut not both. When referring to a disubstituted amino, it is meant thatneither R⁴⁸ nor R⁴⁹ are hydrogen.

“Aminocarbonyl” refers to the group —C(O)NR⁵⁰R⁵¹ where R⁵⁰ and R⁵¹ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aminothiocarbonyl” refers to the group —C(S)NR⁵⁰R⁵¹ where R⁵⁰ and R⁵¹are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aminocarbonylamino” refers to the group —NR⁴⁷C(O)NR⁵⁰R⁵¹ where R⁴⁷ ishydrogen or alkyl and R⁵⁰ and R⁵¹ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic, and where R⁵⁰ and R⁵¹ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminothiocarbonylamino” refers to the group —NR⁴⁷C(S)NR⁵⁰R⁵¹ where R⁴⁷is hydrogen or alkyl and R⁵⁰ and R⁵¹ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R⁵⁰ and R⁵¹ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonyloxy” refers to the group —O—C(O)NR⁵⁰R⁵¹ where R⁵⁰ and R⁵¹are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aminosulfonyl” refers to the group —SO₂NR⁵⁰R⁵¹ where R⁵⁰ and R⁵¹ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aminosulfonyloxy” refers to the group —O—SO₂NR⁵⁰R⁵¹ where R⁵⁰ and R⁵¹are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aminosulfonylamino” refers to the group —NR⁴⁷SO₂NR⁵⁰R⁵¹ where R⁴⁷ ishydrogen or alkyl and R⁵⁰ and R⁵¹ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R⁵⁰ and R⁵¹ are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Amidino” refers to the group —C(═NR⁵²)NR⁵⁰R⁵¹ where R⁵⁰, R⁵¹, and R⁵²are independently selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R⁵⁰ andR⁵¹ are optionally joined together with the nitrogen bound thereto toform a heterocyclic or substituted heterocyclic group, and whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiplecondensed rings (e.g., naphthyl or anthryl) which condensed rings may ormay not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is at an aromatic carbon atom. Preferred aryl groupsinclude phenyl and naphthyl.

“Substituted aryl” refers to aryl groups which are substituted with 1 to5, preferably 1 to 3, or more preferably 1 to 2 substituents selectedfrom the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl,aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy,cycloalkenylthio, substituted cycloalkenylthio, guanidino, substitutedguanidino, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substitutedheteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy,substituted heterocyclyloxy, heterocyclylthio, substitutedheterocyclylthio, nitro, SO₃H, substituted sulfonyl, substitutedsulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are as defined herein.

“Aryloxy” refers to the group —O-aryl, where aryl is as defined herein,that includes, by way of example, phenoxy and naphthoxy.

“Substituted aryloxy” refers to the group —O-(substituted aryl) wheresubstituted aryl is as defined herein.

“Atylthio” refers to the group —S-aryl, where aryl is as defined herein.

“Substituted arylthio” refers to the group —S-(substituted aryl), wheresubstituted aryl is as defined herein.

“Carbonyl” refers to the divalent group —C(O)— which is equivalent to—C(═O)—.

“Carboxyl” or “carboxy” refers to —COOH or salts thereof.

“Carboxyl ester” or “carboxy ester” refers to the groups —C(O)O-alkyl,—C(O)O-substituted alkyl, —C(O)O-alkenyl, —C(O)O-substituted alkenyl,—C(O)O-alkynyl, —C(O)O-substituted alkynyl, —C(O)O-aryl,—C(O)O-substituted aryl, —C(O)O-cycloalkyl, —C(O)O-substitutedcycloalkyl, —C(O)O-cycloalkenyl, —C(O)O-substituted cycloalkenyl,—C(O)O-heteroaryl, —C(O)O-substituted heteroaryl, —C(O)O-heterocyclic,and —C(O)O-substituted heterocyclic wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“(Carboxyl ester)amino” refers to the group —NR⁴⁷C(O)O-alkyl,—NR⁴⁷C(O)O-substituted alkyl, —NR⁴⁷C(O)O-alkenyl, —NR⁴⁷C(O)O-substitutedalkenyl, —NR⁴⁷C(O)O-alkynyl, —NR⁴⁷C(O)O-substituted alkynyl,—NR⁴⁷C(O)O-aryl, —NR⁴⁷C(O)O-substituted aryl, —NR⁴⁷C(O)O-cycloalkyl,—NR⁴⁷C(O)O-substituted cycloalkyl, —NR⁴⁷C(O)O-cycloalkenyl,—NR⁴⁷C(O)O-substituted cycloalkenyl, —NR⁴⁷C(O)O-heteroaryl,—NR⁴⁷C(O)O-substituted heteroaryl, —NR⁴⁷C(O)O-heterocyclic, and—NR⁴⁷C(O)O-substituted heterocyclic wherein R⁴⁷ is alkyl or hydrogen,and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein.

“(Carboxyl ester)oxy” refers to the group —O—C(O)O-alkyl,—O—C(O)O-substituted alkyl, —O—C(O)O-alkenyl, —O—C(O)O-substitutedalkenyl, —O—C(O)O-alkynyl, —O—C(O)O-substituted alkynyl, —O—C(O)O-aryl,—O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl, —O—C(O)O-substitutedcycloalkyl, —O—C(O)O-cycloalkenyl, —O—C(O)O-substituted cycloalkenyl,—O—C(O)O-heteroaryl, —O—C(O)O-substituted heteroaryl,—O—C(O)O-heterocyclic, and —O—C(O)O-substituted heterocyclic whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

A “composition” as used herein, intends an active agent, such as acompound as disclosed herein and a carrier, inert or active. The carriercan be, without limitation, solid such as a bead or resin, or liquid,such as phosphate buffered saline.

Administration or treatment in “combination” refers to administering twoagents such that their pharmacological effects are manifest at the sametime. Combination does not require administration at the same time orsubstantially the same time, although combination can include suchadministrations.

“Cyano” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspino ring systems. The fused ring can be an aryl ring provided that thenon aryl part is joined to the rest of the molecule. Examples ofsuitable cycloalkyl groups include, for instance, adamantyl,cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple cyclic rings and having atleast one >C═C<ring unsaturation and preferably from 1 to 2 sitesof >C═C<ring unsaturation.

“Substituted cycloalkyl” and “substituted cycloalkenyl” refers to acycloalkyl or cycloalkenyl group having from 1 to 5 or preferably 1 to 3substituents selected from the group consisting of oxo, thioxo, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino,substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl,aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy,cycloalkenylthio, substituted cycloalkenylthio, guanidino, substitutedguanidino, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substitutedheteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy,substituted heterocyclyloxy, heterocyclylthio, substitutedheterocyclylthio, nitro, SO₃H, substituted sulfonyl, substitutedsulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are as defined herein.

“Cycloalkyloxy” refers to —O-cycloalkyl.

“Substituted cycloalkyloxy refers to —O-(substituted cycloalkyl).

“Cycloalkylthio” refers to —S-cycloalkyl.

“Substituted cycloalkylthio” refers to —S-(substituted cycloalkyl).

“Cycloalkenyloxy” refers to —O-cycloalkenyl.

“Substituted cycloalkenyloxy” refers to —O-(substituted cycloalkenyl).

“Cycloalkenylthio” refers to —S-cycloalkenyl.

“Substituted cycloalkenylthio” refers to —S-(substituted cycloalkenyl).

“Guanidino” refers to the group —NHC(═NH)NH₂.

“Substituted guanidino” refers to —NR⁵³C(═NR⁵³)N(R⁵³)₂ where each R⁵³ isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclic, andsubstituted heterocyclic and two R⁵³ groups attached to a commonguanidino nitrogen atom are optionally joined together with the nitrogenbound thereto to form a heterocyclic or substituted heterocyclic group,provided that at least one R⁵³ is not hydrogen, and wherein saidsubstituents are as defined herein.

“Halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 10 carbon atomsand 1 to 4 heteroatoms selected from the group consisting of oxygen,nitrogen and sulfur within the ring. Such heteroaryl groups can have asingle ring (e.g., pyridinyl or furyl) or multiple condensed rings(e.g., indolizinyl or benzothienyl) wherein the condensed rings may ormay not be aromatic and/or contain a heteroatom provided that the pointof attachment is through an atom of the aromatic heteroaryl group. Inone embodiment, the nitrogen and/or the sulfur ring atom(s) of theheteroaryl group are optionally oxidized to provide for the N-oxide(N→O), sulfinyl, or sulfonyl moieties. Certain non-limiting examplesinclude pyridinyl, pyrrolyl, indolyl, thiophenyl, oxazolyl, thizolyl,and furanyl.

“Substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to2 substituents selected from the group consisting of the same group ofsubstituents defined for substituted aryl.

“Heteroaryloxy” refers to —O-heteroaryl.

“Substituted heteroaryloxy” refers to the group —O-(substitutedheteroaryl).

“Heteroarylthio” refers to the group —S-heteroaryl.

“Substituted heteroarylthio” refers to the group —S-(substitutedheteroaryl).

“Heterocycle” or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl”refers to a saturated or partially saturated, but not aromatic, grouphaving from 1 to 10 ring carbon atoms and from 1 to 4 ring heteroatomsselected from the group consisting of nitrogen, sulfur, or oxygen.Heterocycle encompasses single ring or multiple condensed rings,including fused bridged and spiro ring systems. In fused ring systems,one or more the rings can be cycloalkyl, aryl, or heteroaryl providedthat the point of attachment is through a non-aromatic ring. In oneembodiment, the nitrogen and/or sulfur atom(s) of the heterocyclic groupare optionally oxidized to provide for the N-oxide, sulfinyl, orsulfonyl moieties.

“Substituted heterocyclic” or “substituted heterocycloalkyl” or“substituted heterocyclyl” refers to heterocyclyl groups that aresubstituted with from 1 to 5 or preferably 1 to 3 of the samesubstituents as defined for substituted cycloalkyl.

“Heterocyclyloxy” refers to the group —O-heterocycyl.

“Substituted heterocyclyloxy” refers to the group —O-(substitutedheterocycyl).

“Heterocyclylthio” refers to the group —S-heterocycyl.

“Substituted heterocyclylthio” refers to the group —S-(substitutedheterocycyl).

Examples of heterocycle and heteroaryls include, but are not limited to,azetidine, pyrrole, furan, thiophene, imidazole, pyrazole, pyridine,pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole,dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine,phenanthroline, isothiazole, phenazine, isoxazolc, phenoxazine,phenothiazine, imidazolidinc, imidazolinc, piperidine, piperazine,indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiomorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,and tetrahydrofuranyl.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O).

Phenylene refers to a divalent aryl ring, where the ring contains 6carbon atoms.

Substituted phenylene refers to phenylenes which are substituted with 1to 4, preferably 1 to 3, or more preferably 1 to 2 substituents selectedfrom the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino,aminocarbonyl, aminothiocarbonyl, aminocarbonylamino,aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl,aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl,aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl,carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano,cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substitutedcycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, cycloalkenyl,substituted cycloalkenyl, cycloalkenyloxy, substituted cycloalkenyloxy,cycloalkenylthio, substituted cycloalkenylthio, guanidino, substitutedguanidino, halo, hydroxy, heteroaryl, substituted heteroaryl,heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substitutedheteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy,substituted heterocyclyloxy, heterocyclylthio, substitutedheterocyclylthio, nitro, SO₃H, substituted sulfonyl, substitutedsulfonyloxy, thioacyl, thiol, alkylthio, and substituted alkylthio,wherein said substituents are as defined herein.

“Spirocycloalkyl” and “spiro ring systems” refers to divalent cyclicgroups from 3 to 10 carbon atoms having a cycloalkyl or heterocycloalkylring with a spiro union (the union formed by a single atom which is theonly common member of the rings) as exemplified by the followingstructure:

“Sulfonyl” refers to the divalent group —S(O)₂—.

“Substituted sulfonyl” refers to the group —SO₂-alkyl, —SO₂-substitutedalkyl, —SO₂-alkenyl, —SO₂-substituted alkenyl, —SO₂-cycloalkyl,—SO₂-substituted cycloalkyl, —SO₂-cycloalkenyl, —SO₂-substitutedcylcoalkenyl, —SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl,—SO₂-substituted heteroaryl, —SO₂-heterocyclic, —SO₂-substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein. Substituted sulfonyl includes groupssuch as methyl-SO₂—, phenyl-SO₂—, and 4-methylphenyl-SO₂—.

“Substituted sulfonyloxy” refers to the group —OSO₂-alkyl,—OSO₂-substituted alkyl, —OSO₂-alkenyl, —OSO₂-substituted alkenyl,—OSO₂-cycloalkyl, —OSO₂-substituted cycloalkyl, —OSO₂-cycloalkenyl,—OSO₂-substituted cylcoalkenyl, —OSO₂-aryl, —OSO₂-substituted aryl,—OSO₂-heteroaryl, —OSO₂-substituted heteroaryl, —OSO₂-heterocyclic,—OSO₂-substituted heterocyclic, wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Thioacyl” refers to the groups H—C(S)—, alkyl-C(S)—, substitutedalkyl-C(S)—, alkenyl-C(S)—, substituted alkenyl-C(S)—, alkynyl-C(S)—,substituted alkynyl-C(S)—, cycloalkyl-C(S)—, substitutedcycloalkyl-C(S)—, cycloalkenyl-C(S)—, substituted cycloalkenyl-C(S)—,aryl-C(S)—, substituted aryl-C(S)—, heteroaryl-C(S)—, substitutedheteroaryl-C(S)—, heterocyclic-C(S)—, and substitutedheterocyclic-C(S)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Thiol” refers to the group —SH.

“Thiocarbonyl” refers to the divalent group —C(S)— which is equivalentto —C(═S)—.

“Thioxo” refers to the atom (═S).

“Alkylthio” refers to the group —S-alkyl wherein alkyl is as definedherein.

“Substituted alkylthio” refers to the group —S-(substituted alkyl)wherein substituted alkyl is as defined herein.

“Optionally substituted” refers to a group selected from that group anda substituted form of that group. Substituted groups are defined herein.In one embodiment, subtituents are selected from C₁-C₁₀ or C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₃-C₈ cycloalkyl, C₂-C₁₀heterocyclyl, C₁-C₁₀ heteroaryl, halo, nitro, cyano, —CO₂H or a C₁-C₆alkyl ester thereof.

“Tautomer” refer to alternate forms of a compound that differ in theposition of a proton, such as enol-keto and imine-enamine tautomers, orthe tautomeric forms of heteroaryl groups containing a ring atomattached to both a ring —NH— moiety and a ring=N— moiety such aspyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles.

“Uracil” as referred to herein has the formula:

“Uracil isostere” refers to an isostere of uracil and does not includeuracil or a halouracil. A halouracil, such as 5-fluorouracil or a uracilcontaining other halogens, are well known to the skilled artisan. Such amoiety provides some or all of the hydrogen bond acceptor-donor-acceptorproperty of uracil and optionally provides other structuralcharacteristics of uracil. A skilled artisan will further appreciate themeaning of this term by reading the non limiting examples of such uracilisosteres provided herein.

As used herein, the term stereochemically pure denotes a compound whichhas 80% or greater by weight of the indicated stereoisomer and 20% orless by weight of other stereoisomers. In a further embodiment, thecompound of Formula (I), (II), or (III) has 90% or greater by weight ofthe stated stereoisomer and 10% or less by weight of otherstereoisomers. In a yet further embodiment, the compound of Formula (I),(II), or (III) has 95% or greater by weight of the stated stereoisomerand 5% or less by weight of other stereoisomers. In a still furtherembodiment, the compound of formula (I), (II), or (III) has 97% orgreater by weight of the stated stereoisomer and 3% or less by weight ofother stereoisomers.

“Pharmaceutically acceptable salt” refers to salts of a compound, whichsalts are suitable for pharmaceutical use and are derived from a varietyof organic and inorganic counter ions well known in the art and include,when the compound contains an acidic functionality, by way of exampleonly, sodium, potassium, calcium, magnesium, ammonium, andtetraalkylammonium; and when the molecule contains a basicfunctionality, salts of organic or inorganic acids, such ashydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, andoxalate (see Stahl and Wermuth, eds., “Handbook of PharmaceuticallyAcceptable Salts,” (2002), Verlag Helvetica Chimica Acta, Zürich,Switzerland), for a discussion of pharmaceutical salts, their selection,preparation, and use.

Generally, pharmaceutically acceptable salts are those salts that retainsubstantially one or more of the desired pharmacological activities ofthe parent compound and which are suitable for in vivo administration.Pharmaceutically acceptable salts include acid addition salts formedwith inorganic acids or organic acids. Inorganic acids suitable forforming pharmaceutically acceptable acid addition salts include, by wayof example and not limitation, hydrohalide acids (e.g., hydrochloricacid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitricacid, phosphoric acid, and the like.

Organic acids suitable for forming pharmaceutically acceptable acidaddition salts include, by way of example and not limitation, aceticacid, trifluoroacetic acid, propionic acid, hexanoic acid,cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid,lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid,3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid,alkylsulfonic acids (e.g., methanesulfonic acid, ethancsulfonic acid,1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, etc.),arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, etc.), glutamic acid, hydroxynaphthoic acid,salicylic acid, stearic acid, muconic acid, and the like.

Pharmaceutically acceptable salts also include salts formed when anacidic proton present in the parent compound is either replaced by ametal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or analuminum ion) or by an ammonium ion (e.g., an ammonium ion derived froman organic base, such as, ethanolamine, diethanolamine, triethanolamine,morpholine, piperidine, dimethylamine, diethylamine, triethylamine, andammonia).

A solvate of a compound is a solid-form of a compound that crystallizeswith less than one, one or more than one molecules of a solvent insidein the crystal lattice. A few examples of solvents that can be used tocreate solvates, such as pharmaceutically acceptable solvates, include,but are not limited to, water, C₁-C₆ alcohols (such as methanol,ethanol, isopropanol, butanol, and can be optionally substituted) ingeneral, tetrahydrofuran, acetone, ethylene glycol, propylene glycol,acetic acid, formic acid, and solvent mixtures thereof. Other suchbiocompatible solvents which may aid in making a pharmaceuticallyacceptable solvate are well known in the art. Additionally, variousorganic and inorganic acids and bases can be added to create a desiredsolvate. Such acids and bases are known in the art. When the solvent iswater, the solvate can be referred to as a hydrate. In some embodiments,one molecule of a compound can form a solvate with from 0.1 to 5molecules of a solvent, such as 0.5 molecules of a solvent (hemisolvate,such as hemihydrate), one molecule of a solvent (monosolvate, such asmonohydrate) and 2 molecules of a solvent (disolvate, such asdihydrate).

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages. Such delivery is dependent ona number of variables including the time period for which the individualdosage unit is to be used, the bioavailability of the therapeutic agent,the route of administration, etc. It is understood, however, thatspecific dose levels of the therapeutic agents disclosed herein for anyparticular subject depends upon a variety of factors including theactivity of the specific compound employed, bioavailability of thecompound, the route of administration, the age of the animal and itsbody weight, general health, sex, the diet of the animal, the time ofadministration, the rate of excretion, the drug combination, and theseverity of the particular disorder being treated and form ofadministration. In general, one will desire to administer an amount ofthe compound that is effective to achieve a serum level commensuratewith the concentrations found to be effective in vivo. Theseconsiderations, as well as effective formulations and administrationprocedures are well known in the art and are described in standardtextbooks. Consistent with this definition and as used herein, the term“therapeutically effective amount” is an amount sufficient to treat aspecified disorder or disease or alternatively to obtain apharmacological response such as inhibiting dUTPase.

As used herein, “treating” or “treatment” of a disease in a patientrefers to (1) preventing the symptoms or disease from occurring in ananimal that is predisposed or does not yet display symptoms of thedisease; (2) inhibiting the disease or arresting its development; or (3)ameliorating or causing regression of the disease or the symptoms of thedisease. As understood in the art, “treatment” is an approach forobtaining beneficial or desired results, including clinical results. Forthe purposes of this technology, beneficial or desired results caninclude one or more, but are not limited to, alleviation or ameliorationof one or more symptoms, diminishment of extent of a condition(including a disease), stabilized (i.e., not worsening) state of acondition (including disease), delay or slowing of condition (includingdisease), progression, amelioration or palliation of the condition(including disease), states and remission (whether partial or total),whether detectable or undetectable.

“dUTPase” means any of the following, which are considered to besynonymous, “deoxyuridine triphosphate nucleotidohydrolase”,“deoxyuridine triphosphate pyrophosphatase”, “dUTP nucleotidohydrolasc”,“dUTP pyrophosphatase”, and other equivalent nomenclature for thedUTPase enzyme. In one aspect, dUTPase intends DUT-N and DUT-M. In otheraspects, it is DUT-N only, or alternatively, DUT-M only. The amino acidand coding sequences for dUTPase are known in the art and disclosed inU.S. Pat. No. 5,962,246. Methods for expressing and screening forexpression level of the enzyme are disclosed in U.S. Pat. No. 5,962,246and Ladner et al. (US Patent Publ. No. 2011/0212467A1).

“DUT-N” means the nuclear form of dUTPase.

“DUT-M” means the mitochondrial or cytoplasmic form of dUTPase.

“dUTPase-directed therapy” intends therapeutics that target the dUTPasepathway, e.g., in the case of cancer, e.g. TS-directed therapies and thefluoropyrimidines (such as 5-FU), pemetrexed (Alimta®), capecitabine(Xcloda®), S-1 and antifolatcs (such as methotrexate) and chemicalequivalents thereof. Non-limiting examples include 5-flurouracil (5-FU),TS-directed therapies and 5-FU based adjuvant therapy. Combinationtherapies can include any intervention that alters nucleotide poolsand/or sensitizes the immune cells or viruses to the dUTPase inhibitor,as are well known to the skilled artisan. For rheumatoid arthritis, forexample, the combination can be with an dihydrofolate reductase (DHFR)inhibitor such as methotrexate.

5-fluorouracil (5-FU) belongs to the family of therapy drugs calledpyrimidine based anti-metabolites. It is a pyrimidine analog, which istransformed into different cytotoxic metabolites that are thenincorporated into DNA and RNA thereby inducing cell cycle arrest andapoptosis. Chemical equivalents are pyrimidine analogs which result indisruption of DNA replication. Chemical equivalents inhibit cell cycleprogression at S phase resulting in the disruption of cell cycle andconsequently apoptosis. Equivalents to 5-FU include prodrugs, analogsand derivative thereof such as 5′-deoxy-5-fluorouridine(doxifluoroidine), 1-tetrahydrofuranyl-5-fluorouracil (ftorafur),capecitabine (Xeloda®), S-1 (MBMS-247616, consisting of tegafur and twomodulators, a 5-chloro-2,4-dihydroxypyridine and potassium oxonate),ralititrexed (tomudex), nolatrexed (Thymitaq, AG337), LY231514 andZD9331, as described for example in Papamicheal (1999) The Oncologist4:478-487.

“5-FU based adjuvant therapy” refers to 5-FU alone or alternatively thecombination of 5-FU with other treatments, that include, but are notlimited to radiation, methyl-CCNU, leucovorin, oxaliplatin, irinotecin,mitomycin, cytarabine, levamisole. Specific treatment adjuvant regimensare known in the art as FOLFOX, FOLFOX4, FOLFIRI, MOF (semustine(methyl-CCNU), vincrisine (Oncovin®) and 5-FU). For a review of thesetherapies see Beaven and Goldberg (2006) Oncology 20(5):461-470. Anexample of such is an effective amount of 5-FU and Leucovorin. Otherchemotherapeutics can be added, e.g., oxaliplatin or irinote can.

Capecitabine is a prodrug of (5-FU) that is converted to its active formby the tumor-specific enzyme PynPase following a pathway of threeenzymatic steps and two intermediary metabolites,5′-deoxy-5-fluorocytidine (5′-DFCR) and 5′-deoxy-5-fluorouridine(5′-DFUR). Capecitabine is marketed by Roche under the trade nameXeloda®.

Leucovorin (Folinic acid) is an adjuvant used in cancer therapy. It isused in synergistic combination with 5-FU to improve efficacy of thechemotherapeutic agent. Without being bound by theory, addition ofLeucovorin is believed to enhance efficacy of 5-FU by inhibitingthymidylate synthase. It has been used as an antidote to protect normalcells from high doses of the anticancer drug methotrexate and toincrease the antitumor effects of fluorouracil (5-FU) andtegafur-uracil. It is also known as citrovorum factor and Wellcovorin.This compound has the chemical designation of L-Glutamic acidN[4[[(2-amino-5-formyl1,4,5,6,7,8hexahydro4oxo6-pteridinyl)methyl]amino]b-enoyl],calcium salt (1:1).

“Oxaliplatin” (Eloxatin) is a platinum-based chemotherapy drug in thesame family as cisplatin and carboplatin. It is typically administeredin combination with fluorouracil and leucovorin in a combination knownas FOLFOX for the treatment of colorectal cancer. Compared to cisplatin,the two amine groups are replaced by cyclohexyldiamine for improvedantitumour activity. The chlorine ligands are replaced by the oxalatobidentate derived from oxalic acid in order to improve water solubility.Equivalents to Oxaliplatin are known in the art and include, but are notlimited to cisplatin, carboplatin, aroplatin, lobaplatin, nedaplatin,and JM-216 (see McKeage et al. (1997) J. Clin. Oncol. 201:1232-1237 andin general, Chemotherapy for Gynecological Neoplasm, Curr. Therapy andNovel Approaches, in the Series Basic and Clinical Oncology, Angioli etal. Eds., 2004).

“FOLFOX” is an abbreviation for a type of combination therapy that isused to treat cancer. This therapy includes 5-FU, oxaliplatin andleucovorin. “FOLFIRI” is an abbreviation for a type of combinationtherapy that is used treat cancer and comprises, or alternativelyconsists essentially of, or yet further consists of 5-FU, leucovorin,and irinotecan. Information regarding these treatments are available onthe National Cancer Institute's web site, cancer.gov, last accessed onJan. 16, 2008.

Irinotecan (CPT-11) is sold under the trade name of Camptosar. It is asemi-synthetic analogue of the alkaloid camptothecin, which is activatedby hydrolysis to SN-38 and targets topoisomerase I. Chemical equivalentsare those that inhibit the interaction of topoisomerase I and DNA toform a catalytically active topoisomerase I-DNA complex. Chemicalequivalents inhibit cell cycle progression at G2-M phase resulting inthe disruption of cell proliferation.

The term “adjuvant” therapy refers to administration of a therapy orchemotherapeutic regimen to a patient after removal of a tumor bysurgery. Adjuvant therapy is typically given to minimize or prevent apossible cancer reoccurrence. Alternatively, “neoadjuvant” therapyrefers to administration of therapy or chemotherapeutic regimen beforesurgery, typically in an attempt to shrink the tumor prior to a surgicalprocedure to minimize the extent of tissue removed during the procedure.

The phrase “first line” or “second line” or “third line” etc., refers tothe order of treatment received by a patient. First line therapyregimens are treatments given first, whereas second or third linetherapy are given after the first line therapy or after the second linetherapy, respectively. The National Cancer Institute defines first linetherapy as “the first treatment for a disease or condition. In patientswith cancer, primary treatment can be surgery, chemotherapy, radiationtherapy, or a combination of these therapies. First line therapy is alsoreferred to those skilled in the art as primary therapy and primarytreatment.” See National Cancer Institute website as www.cancer.gov,last visited on May 1, 2008. Typically, a patient is given a subsequentchemotherapy regimen because the patient did not shown a positiveclinical or sub-clinical response to the first line therapy or the firstline therapy has stopped.

As used herein, the term “antifolate” intends a drug or biologic thatimpairs the function of folic acids, e.g., an antimetabolite agent thatinhibits the use of a metabolite, i.e. another chemical that is part ofnormal metabolism. In cancer treatment, antimetabolites interfere withDNA production, thus cell division and growth of the tumor. Non-limitingexamples of these agents are dihydrofolate reductase inhibitors, such asmethotrexate, Aminopterin, and Pemetrexed; thymidylate synthaseinhibitors, such as Raltitrexed or Pemetrexed; purine based, i.e. anadenosine deaminase inhibitor, such as Pentostatin, a thiopurine, suchas Thioguanine and Mercaptopurine, a halogenated/ribonucleotidereductase inhibitor, such as Cladribine, Clofarabine, Fludarabine, or aguanine/guanosine: thiopurine, such as Thioguanine; or Pyrimidine based,i.e. cytosine/cytidine: hypomethylating agent, such as Azacitidinc andDccitabinc, a DNA polymcrasc inhibitor, such as Cytarabine, aribonucleotide reductase inhibitor, such as Gemcitabine, or athymine/thymidine: thymidylate synthase inhibitor, such as aFluorouracil (5-FU).

In one aspect, the term “chemical equivalent” means the ability of thechemical to selectively interact with its target protein, DNA, RNA orfragment thereof as measured by the inactivation of the target protein,incorporation of the chemical into the DNA or RNA or other suitablemethods. Chemical equivalents include, but are not limited to, thoseagents with the same or similar biological activity and include, withoutlimitation a pharmaceutically acceptable salt, and/or a solvate thereof,or mixtures thereof that interact with and/or inactivate the same targetprotein, DNA, or RNA as the reference chemical.

The terms “oligonucleotide” or “polynucleotide” or “portion,” or“segment” thereof refer to a stretch of polynucleotide residues which islong enough to use in PCR or various hybridization procedures toidentify or amplify identical or related parts of mRNA or DNA molecules.The polynucleotide compositions of this invention include RNA, cDNA,genomic DNA, synthetic forms, and mixed polymers, both sense andantisense strands, and may be chemically or biochemically modified ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those skilled in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties(e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.),chelators, alkylators, and modified linkages (e.g., alpha anomericnucleic acids, etc.). Also included are synthetic molecules that mimicpolynucleotides in their ability to bind to a designated sequence viahydrogen bonding and other chemical interactions. Such molecules areknown in the art and include, for example, those in which peptidelinkages substitute for phosphate linkages in the backbone of themolecule.

When a genetic marker, e.g., over expression of dUTPase, is used as abasis for selecting a patient for a treatment described herein, thegenetic marker is measured before and/or during treatment, and thevalues obtained are used by a clinician in assessing any of thefollowing: (a) probable or likely suitability of an individual toinitially receive treatment(s); (b) probable or likely unsuitability ofan individual to initially receive treatment(s); (c) responsiveness totreatment; (d) probable or likely suitability of an individual tocontinue to receive treatment(s); (e) probable or likely unsuitabilityof an individual to continue to receive treatment(s); (f) adjustingdosage; (g) predicting likelihood of clinical benefits; or (h) toxicity.As would be well understood by one in the art, measurement of thegenetic marker in a clinical setting is a clear indication that thisparameter was used as a basis for initiating, continuing, adjustingand/or ceasing administration of the treatments described herein.

“Cancer” is a known medically as a malignant neoplasm, is a broad groupof diseases involving unregulated cell growth. In cancer, cells divideand grow uncontrollably, forming malignant tumors, and invade nearbyparts of the body. Non-limiting examples include colon cancer,colorectal cancer, gastric cancer, esophogeal cancer, head and neckcancer, breast cancer, lung cancer, stomach cancer, liver cancer, gallbladder cancer, or pancreatic cancer or leukemia.

The following are non-limiting aspects of the present disclosure.

Compounds

In one embodiment, provided herein is a compound of Formula (I), whichis of Formula (III):

-   wherein A is an uracil isostere of formula:

-   each V is independently O or S,-   R¹¹ is hydrogen, halo, R¹² or —O—R¹², wherein R¹² is C₁-C₆ alkyl,    C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted with 1-3    hydroxy, fluoro, chloro, and amino substituent,-   r is 1, 2, or 3,-   Y¹⁰ is O, S, SO, SO₂, NH or NR¹⁵, or L¹ is joined at Y¹⁰ with Y¹⁰    being a nitrogen atom;-   R¹⁵ is C₁-C₆ alkyl optionally substituted with 1-3 C alkoxy,    hydroxy, amino, and oxo groups,-   L¹- is

-   Y¹ is CH₂, O, S,-   X¹⁰ is NH, NCO₂R²⁰, O, —CO—, —CO—NH—, or CH₂,-   R²⁰ is C₁-C₆alkyl optionally substituted with 1-3 C₆-C₁₀ aryl    groups,-   u is 0, 1, 2, 3, or 4,-   R^(z) is hydroxy or hydrogen,-   R^(w) is C₁-C₆ alkyl or hydrogen, and-   the phenylene and the heteroarylene rings are optionally    substituted,-   Z is phenyl or a 5 or 6 member heteroaryl substituted with an R⁶ and    an R⁶⁰ groups, wherein the R⁶ and the R⁶⁰ are positioned 1,2 with    respect to each other,-   R⁶ is hydrogen, optionally substituted C₁-C₆ alkoxy, or halo, and-   R⁶⁰ is —OR⁷ or —NHR⁷R⁷⁰,-   R⁷ is optionally substituted C₁-C₁₀ alkyl, optionally substituted    C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl, optionally    substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₁₀    heteroaryl, optionally substituted C₃-C₁₀ heterocyclyl, or    optionally substituted phenyl, and-   R⁷⁰ is hydrogen or R⁷.    In one embodiment, each V is O. In another embodiment, each V is S.    In another embodiment, X¹⁰ is NH. In another embodiment, X¹⁰ is    NCO₂R²⁰. In another embodiment, X¹⁰ is O. In another embodiment, X¹⁰    is —CO—. In another embodiment, X¹⁰ is —CO—NH—. In another    embodiment, X¹⁰ is CH₂.

In another embodiment, the uracil isostere is:

wherein Y¹⁰ is O, S, SO, SO₂, NH or NR¹⁵.

In some embodiments, the uracil isostere is:

In another embodiment, Y¹⁰ is O. In another embodiment, Y¹⁰ is NH.

In some embodiments, the uracil isostere is:

In another embodiment, R¹¹ is hydrogen. In another embodiment, R¹¹ isC₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionally substituted with1-3 hydroxy, fluoro, chloro, and amino substituent. In anotherembodiment, R¹¹ is methyl. In another embodiment, R¹ is a non-hydrogensubstituent, and r is 1. In another embodiment, R¹ is a non-hydrogensubstituent, and r is 2. In another embodiment, R¹ is a non-hydrogensubstituent, and r is 3.

In another embodiment, —W—X—Y— is —CH₂—X—SO₂—NH—CH(R^(Y))—;—CH₂—X—SO₂—NH—C(R^(Y))₂—; or —CH₂—X—B—CH₂CR^(X)R^(W)—,

-   X is optionally substituted C₁-C₆ alkylene wherein one of the    methylene groups within the alkylene chain is optionally replaced    with an O or S atom, such that X is optionally substituted alkylene    or optionally substituted heteroalkylene;-   B is a optionally substituted C₃-C₁₀ heteroaryl;-   R^(Y) an R^(W) are independently hydrogen or C₁-C₆ alkyl; and-   R^(z) is hydrogen or hydroxy.

In one embodiment, B is a 5 membered heteroaryl containing up to 3 or 4heteroatoms selected from nitrogen, sulfur and oxygen. In oneembodiment, B is:

In another embodiment, —W—X—Y— or L¹ is

Y¹ is CH₂, O or S,

-   X¹⁰ and u are as defined herein,-   R^(z) is hydroxy or hydrogen,-   R^(w) is C₁-C₆ alkyl or hydrogen,-   the phenylene and the heteroarylene rings are optionally    substituted.

In some embodiments, —W—X—Y— or L¹ is

In some embodiments, —W—X—Y— or L¹ is

In another embodiment, R⁴ is optionally substituted C₆-C₁₀ aryl. Inanother embodiment, R⁴ is optionally substituted C₂-C₁₀ heterocyclicgroup. In another embodiment, R⁴ is optionally substituted C₁-C₁₀heteroaryl group. In another embodiment, when Y is -L¹⁰-B¹-L¹¹-, Z isR⁴.

In some embodiments, Z is phenyl or a 5 or 6 membered heteroarylsubstituted with an R⁶ and an R⁶⁰ groups, wherein the R⁶ and the R⁶⁰ arepositioned 1,2 with respect to each other,

-   R⁶ is hydrogen, optionally substituted C₁-C₆ alkoxy, or halo,-   R⁶⁰ is —OR⁷ or —NHR⁷R⁷⁰,-   R⁷ is optionally substituted C₁-C₆ alkyl, optionally substituted    C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl, optionally    substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₁₀    heteroaryl, optionally substituted C₃-C₁₀ heterocyclyl, or    optionally substituted phenyl, and-   R⁷⁰ is hydrogen or R⁷.

In some embodiments, Z or R⁴ is selected from:

-   wherein each R⁶ and R⁷ independently are defined as in any aspect or    embodiment above,-   each R⁶¹ and R⁶² independently is N or CH, provided that at least    one of R⁶¹ and R⁶² is N,-   each R⁶³ independently is NR⁷⁰, S, O, and-   each R⁶⁴ independently is N or CH.

In some embodiments, provided herein is a compound of formula:

wherein the variables are as defined herein.

In some embodiments, provided herein is a compound of formula:

wherein the variables are as defined herein, and R⁸ is defined as R⁷,independently of each other.

In another embodiment, Z is:

-   R⁶ is hydrogen, optionally substituted C₁-C₆ alkoxy, or halo, and-   R⁷ is optionally substituted C₁-C₆ alkyl, optionally substituted    C₂-C₆ alkenyl, optionally substituted C₂-C₆ alkynyl, optionally    substituted C₃-C₈ cycloalkyl, optionally substituted C₃-C₁₀    heteroaryl, optionally substituted C₃-C₁₀ heterocyclyl, or    optionally substituted phenyl.

In one embodiment, R⁶ is hydrogen. In one embodiment, R⁶ is halo. Inanother embodiment, R⁶ is fluoro. In one embodiment, R⁶ is C₁-C₆ alkoxy.In one embodiment, R⁶ is C₁-C₆ alkoxy substituted with 1-3 fluorogroups. In some embodiments, R⁶ is hydrogen, F, Cl, OMe, or OCF₃.

In one embodiment, R⁷ is C₁-C₆ alkyl substituted with a C₃-C₈cycloalkyl, C₂-C₁₀ heterocyclyl, or C₁-C₁₀ heteroaryl. In oneembodiment, R⁷ is

In one embodiment, R₇ is C₁-C₆ alkyl optionally substituted with a C₃-C₈cycloalkyl, 4-8 membered heterocyclyl, or R⁷ is C₁-C₆ alkyl substitutewith 1-3 fluoro atoms.

In another embodiment, R⁷ is:

wherein t is 1, 2, or 3. In another embodiment, t is 1. In anotherembodiment, t is 2. In another embodiment, t is 3.

In another embodiment, the cycloalkyl is cyclopropyl. In anotherembodiment, the cycloalkyl is cyclobutyl. In another embodiment, thecycloalkyl is cyclopentyl. In another embodiment, the cycloalkyl iscyclohexyl. In another embodiment, R⁷ is isobutyl. In anotherembodiment, R⁷ is neopentyl.

In another embodiment, the heterocyclyl is

In another embodiment, the heterocyclyl is:

In another embodiment, the heterocyclyl is:

In another embodiment, the compound is of formula:

wherein L₁ is defined as above.

In another embodiment, the compound is of formula:

wherein L₁ is defined as above.

In another embodiment, the compound is of formula:

wherein L₁ is defined as above.

In another embodiment, the compound is of formula:

wherein L₁ is defined as above.

In one embodiment, provided herein is a compound of formula:

wherein A is selected from:

-   wherein Y¹⁰, R¹¹ and r are as defined herein;-   X¹⁰ is NH, NCO₂R²⁰, O, —CO—NH—, or CH₂;-   R²⁰ is C₁-C₆ alkyl optionally substituted with 1-3 C₆-C₁₀ aryl    groups;-   u is 0, 1, 2, 3, or 4;-   R¹¹ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl    wherein each alkyl, alkenyl, and alkynyl is optionally substituted    with 1-3 hydroxy, fluoro, chloro, and amino substituent; and-   r is 1 or 2.

In another embodiment, X¹⁰ is CH₂. In another embodiment, X¹⁰ is NH. Inanother embodiment, X¹⁰ is —CO—NH—. In another embodiment, u is 1. Inanother embodiment, u is 2. In another embodiment, u is 3.

In another embodiment, provided herein is a compound selected from:

and a diastereomer or an enantiomer thereof.

The compounds provided herein include individual, separated enantiomersand diastereomers that are stereochemically pure or enriched,tautotomers, and pharmaceutically acceptable salts, and/or a solvatethereof, wherever applicable. As used herein, the term stereochemicallypure denotes a compound which has 80% or greater by weight of theindicated stereoisomer and 20% or less by weight of other stereoisomers.In a further aspect, the compounds as described herein have 90% orgreater by weight of the denoted stereoisomer and 10% or less by weightof other stereoisomers. In a yet further embodiment, the compounds ofthis disclosure have 95% or greater by weight of the denotedstereoisomer and 5% or less by weight of other stereoisomers. In a stillfurther embodiment, the compounds have 97% or greater by weight of thedenoted stereoisomer and 3% or less by weight of other stereoisomers.Any one or more of the compounds can be provided as compositions, e.g.,of pharmaceutically acceptable salt, and/or a solvate thereof.

Synthesis

The following general synthetic scheme is used to prepare the compoundsprovided herein. For example, compounds of formula I are synthesized asshown in the reaction scheme below:

wherein A, X—Y—, L₁, and Z are as defined herein, and H is hydrogen. Ingeneral, uracil, uracil isostere, or a halo uracil is treated with asuitable base such as butyl lithium in a solvent such as tetrahydrofuranor dimethylformamide. The A(−) anion obtained by deprotonation of theA-H moiety and also by halogen exchange of an A-halo bond with an alkyllithium. It is then coupled with compound B, wherein LG is a leavinggroup such as halogen, tosylate or mesylate to provide compounds offormula (I). In some embodiments, protection of an NH, OH, or such othergroup in uracil, uracil isostere, halo uracil, or the —W—X—Y—Z moiety isrequired. Compounds of formula (III) can also be synthesized in ananalogous manner. An uracil isostere containing an —NH— or an NH₂ groupcan also be alkylated following reductive amination as is well known toa skilled artisan.

A-ring substituted compounds provided here are synthesized as shownbelow and or following methods well known in the art in view of thepresent disclosure. See also, Journal of Heterocyclic Chemistry (2005)vol. 42, #2 p. 201-207, Journal of the American Chemical Society (2009)vol. 131, p. 8196-8210, Journal of Heterocyclic Chemistry (1994) vol.31, #2 p. 565-568, and Journal of Medicinal Chemistry (1994) vol. 37,#13 p. 2059-2070, each of which is incorporated herein by reference.

Additional —W—X—Y—Z moieties are disclosed in US 2011/0082163; US2012/0225838; Miyahara et al., J. Med. Chem. (2012) 55, 2970-2980;Miyakoshi et al., J. Med. Chem. (2012) 55, 2960-2969; Miyahara et al.,J. Med. Chem. (2012) 55 (11), pp 5483-5496; and Miyakoshi et al., J.Med. Chem. (2012) 55 (14), pp 6427-6437 (each of which are incorporatedherein by reference) and can be used with the A moieties disclosedherein.

These and other compounds provided herein are synthesized following artrecognized methods with the appropriate substitution of commerciallyavailable reagents as needed. For example, and without limitation,methods for synthesizing certain other compounds are described in US2011/0082163; US 2012/0225838; Miyahara et al., J. Med. Chem. (2012) 55,2970-2980; Miyakoshi et al., J. Med. Chem. (2012) 55, 2960-2969;Miyahara et al., J. Med. Chem. (2012) 55 (11), pp 5483-5496; andMiyakoshi et al., J. Med. Chem. (2012) 55 (14), pp 6427-6437 (eachsupra), which methods can be adapted by the skilled artisan upon readingthis disclosure and/or based on synthetic methods well known in the art,to prepare the compounds provided herein. Protection deprotectionmethods and protecting groups useful for such purposes are well known inthe art, for example in Greene's Protective Groups in Organic Synthesis,4^(th) Edition, Wiley, 2006, or a later edition of the book.

The compounds and the intermediates are separated from the reactionmixture, when desired, following art known methods such ascrystallization, chromatography, distillation, and the like. Thecompounds and the intermediates are characterized by art known methodssuch as thin layer chromatography, nuclear magnetic resonancespectroscopy, high performance liquid chromatography, and the like. Asdescribed in detail herein, a racemic or diastereomeric mixture of thecompound can be separated or enriched to the enantiomers anddiastereomers and tested and used diagnostically or therapeutically asdescribed herein.

Methods of testing and using the compounds provided herein are performedfollowing art recognized in vitro (cell free), ex vivo or in vivomethods. For example, and without limitation, certain methods fortesting and using other compounds are described in US 2011/0082163; US2012/0225838; Miyahara et al., J. Med. Chem. (2012) 55, 2970-2980;Miyakoshi et al., J. Med. Chem. (2012) 55, 2960-2969; Miyahara et al.,J. Med. Chem. (2012) 55 (11), pp 5483-5496; Miyakoshi et al., J. Med.Chem. (2012) 55 (14), pp 6427-6437 (each of which in incorporated byreference), which methods can be adapted by the skilled artisan uponreading this disclosure and/or based on methods well known in the art,to test and use the compounds provided herein.

Compositions

Compositions, including pharmaceutical compositions comprising thecompounds described herein can be manufactured by means of conventionalmixing, dissolving, granulating, dragee-making levigating, emulsifying,encapsulating, entrapping, or lyophilization processes. The compositionscan be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients, orauxiliaries which facilitate processing of the compounds provided hereininto preparations which can be used pharmaceutically.

The compounds of the technology can be administered by parenteral (e.g.,intramuscular, intraperitoneal, intravenous, ICV, intracisternalinjection or infusion, subcutaneous injection, or implant), oral, byinhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g.,urethral suppository) or topical routes of administration (e.g., gel,ointment, cream, aerosol, etc.) and can be formulated, alone ortogether, in suitable dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants, excipients,and vehicles appropriate for each route of administration.

In one embodiment, this disclosure relates to a composition comprising acompound as described herein and a carrier.

In another embodiment, this disclosure relates to a pharmaceuticalcomposition comprising a compound as described herein and apharmaceutically acceptable carrier.

In another embodiment, this disclosure relates to a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundas described herein and a pharmaceutically acceptable carrier.

The pharmaceutical compositions for the administration of the compoundscan be conveniently presented in dosage unit form and can be prepared byany of the methods well known in the art of pharmacy. The pharmaceuticalcompositions can be, for example, prepared by uniformly and intimatelybringing the compounds provided herein into association with a liquidcarrier, a finely divided solid carrier or both, and then, if necessary,shaping the product into the desired formulation. In the pharmaceuticalcomposition the compound provided herein is included in an amountsufficient to produce the desired therapeutic effect. For example,pharmaceutical compositions of this disclsoure may take a form suitablefor virtually any mode of administration, including, for example,topical, ocular, oral, buccal, systemic, nasal, injection, infusion,transdermal, rectal, and vaginal, or a form suitable for administrationby inhalation or insufflation.

For topical administration, the compounds can be foiuiulated assolutions, gels, ointments, creams, suspensions, etc., as is well-knownin the art.

Systemic formulations include those designed for administration byinjection (e.g., subcutaneous, intravenous, infusion, intramuscular,intrathecal, or intraperitoneal injection) as well as those designed fortransdermal, transmucosal, oral, or pulmonary administration.

Useful injectable preparations include sterile suspensions, solutions,or emulsions of the compounds provided herein in aqueous or oilyvehicles. The compositions may also contain formulating agents, such assuspending, stabilizing, and/or dispersing agents. The formulations forinjection can be presented in unit dosage form, e.g., in ampules or inmultidose containers, and may contain added preservatives.

Alternatively, the injectable formulation can be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, and dextrose solution, before use.To this end, the compounds provided herein can be dried by any art-knowntechnique, such as lyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants are knownin the art.

For oral administration, the pharmaceutical compositions may take theform of, for example, lozenges, tablets, or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone,or hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose, or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc, or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). The tablets can be coated by methods well known in theart with, for example, sugars, films, or enteric coatings.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions, and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents, and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain thecompounds provided herein in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients can be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents (e.g., corn starch oralginic acid); binding agents (e.g. starch, gelatin, or acacia); andlubricating agents (e.g., magnesium stearate, stearic acid, or talc).The tablets can be left uncoated or they can be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate can be employed. They may also becoated by the techniques well known to the skilled artisan. Thepharmaceutical compositions of the technology may also be in the form ofoil-in-water emulsions.

Liquid preparations for oral administration may take the form of, forexample, elixirs, solutions, syrups, or suspensions, or they can bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations can be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives, orhydrogenated edible fats); emulsifying agents (e.g., lecithin, oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol, Cremophore™, or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, preservatives, flavoring,coloring, and sweetening agents as appropriate.

Use of Compounds for Preparing Medicaments

The compounds and compositions of the present invention are also usefulin the preparation of medicaments to treat a variety of pathologies asdescribed herein. The methods and techniques for preparing medicamentsof a composition are known in the art. For the purpose of illustrationonly, pharmaceutical formulations and routes of delivery are detailedherein.

Thus, one of skill in the art would readily appreciate that any one ormore of the compositions described above, including the many specificembodiments, can be used by applying standard pharmaceuticalmanufacturing procedures to prepare medicaments to treat the manydisorders described herein. Such medicaments can be delivered to thesubject by using delivery methods known in the pharmaceutical arts.

Methods and Therapies

The compositions and compounds as disclosed herein are useful in methodsof inhibiting dUTPase or enhancing the efficacy of a dUTPase-directedtherapy, or yet further, reversing resistance to dUTPase therapies. Themethods comprise, or alternatively consist essentially of, or yetfurther consist of, contacting the dUTPase with an effective amount ofthe compound or composition as disclosed herein. In one embodiment, themethods further comprise, or alternatively consist essentially of, oryet further consist of, contacting the dUTPase with an effective amountof a dUTPase-directed therapy. In one aspect, the contacting of thedUTPase-directed therapy is prior to, concurrent or subsequent tocontacting with the compound or composition of this disclosure.

One of skill in the art can also determine if the compound orcombination inhibits dUTPase in vitro by contacting the compound orcombination with purified or recombinant dUTPase in a cell free system.The purified or recombinant dUTPase and can be from any species, e.g.,simian, canine, bovine, ovine, rat, mouse or human. In one aspect, thedUTPase is DUT-N or DUT-M. Isolation, characterization and expression ofdUTPase isoforms are disclosed in U.S. Pat. No. 5,962,246 and known inthe art.

The contacting can be performed cell-free in vitro or cx vivo with acell or in a cell culture. When performed in vitro or ex vivo, thecompounds, compositions or agents can be directly added to the enzymesolution or added to the cell culture medium. When practiced in vitro orex vivo, the method can be used to screen for novel combinationtherapies, formulations or treatment regimens, prior to administrationto administration to an animal or a human patient. Methods to quantifyinhibition are known in the art, see, U.S. Patent Publ. Nos.2010/0075924 and 2011/0212467 and U.S. Pat. No. 7,601,702. For example,a fixed dose of a dUTPase directed therapy (e.g., 5-FU or Pemetrexed)can be added to the system and varying amounts of the compound can besubsequently added to system. Alternatively, a fixed dose of a compoundof this invention can be added to the system and varying amounts of thedUTPase directed therapy (e.g., 5-FU or Pemetrexed) compound can besubsequently added to system.

In one aspect, the contacting is ex vivo and the cell or tissue to becontacted over expresses dUTPase. These cells can be isolated from apatient prior to administration to the patient or can be purchased froma depository such as the American Type Culture Collection (ATCC).Non-limiting examples of animal (e.g., canine, an equine, a bovine, afeline, an ovine, a mouse, a rat or a simian) and human cells that areknown to over express dUTPase include, without limitation cancer cells,e.g. colon cancer, colorectal cancer, gastric cancer, head and neckcancer, breast cancer, stomach cancer or lung cancer. The cancer can bemetastatic or non-metastatic. Methods to quantify inhibition are knownin the art, see, U.S. Patent Publ. Nos. 2010/0075924 and 2011/0212467and U.S. Pat. No. 7,601,702 and Wilson et al. (2012) Mol. Cancer Ther.11:616-628.

When practiced in vivo in a patient such as an animal or human, thecompounds, compositions or agents are administered in an effectiveamount by a suitable route of administration, as determined by atreating physician taking into account the patient, disease and otherfactors. When practiced in a non-human animal, e.g., an appropriatemouse model, the method can be used to screen for novel combinationtherapies, formulations or treatment regimens, prior to administrationto a human patient.

This disclosure also provides methods of treating a disease whosetreatment is impeded by the expression of dUTPase, comprising, oralternatively consisting essentially of, or yet further consisting of,administering to a patient in need of such treatment an effective amountof the compound or composition of this disclosure, thereby treating thedisease. In one aspect, the method further comprises isolating a cell ortissue sample from the patient and screening for the expression level ofdUTPase, wherein over expression of dUTPase in the sample as compared toa control sample serves as a basis for selecting the patient as suitablefor the method and therapies. Methods to quantify dUTPase are known inthe art. Effective amounts will vary with the patient, the disease andthe general health of the patient and are determined by the treatingphysician. Methods to quantify inhibition are known in the art, see,U.S. Patent Publ. Nos. 2010/0075924 and 2011/0212467 and U.S. Pat. No.7,601,702 and Wilson et al. (2012) Mol. Cancer Ther. 11:616-628. If thepatient sample shows over expression of dUTPase, the therapy isadministered to the patient. If the patient sample does not show overexpression, an alternate therapy is chosen. The screen can be repeatedthroughout therapy as a means to monitor the therapy and/or dosageregimen.

To practice this method, the sample is a patient sample containing thetumor tissue, normal tissue adjacent to said tumor, normal tissue distalto said tumor or peripheral blood lymphocytes. In a further aspect, thepatient or patient population to be treated also is treatment naïve.

In one aspect, the method also requires isolating a sample containingthe marker from the patient to be treated. It is conceivable that one ofskill in the art will be able to analyze and identify markers in situ atsome point in the future. Accordingly, in one aspect, the inventions ofthis application are not to be limited to requiring isolation of thepatient sample prior to analysis.

These methods also are not limited by the technique that is used toidentify the expression level of dUTPase or other relevant enzyme ormarkers. Suitable methods include but are not limited to the use ofhybridization probes, antibodies, primers for PCR analysis, and genechips, slides and software for high throughput analysis. Additionalmarkers can be assayed and used as negative controls.

In one aspect, the subject or patient is an animal or a human patient.Non-limiting examples of animals include a feline, a canine, a bovine,an equine, an ovine, a mouse, a rat or a simian.

Diseases in which treatment is impeded by the expression of dUTPaseinclude, without limitation, cancer, viral infection, bacterialinfection or an autoimmune disorder. For example, in rheumatoidarthritis, inflammatory bowel disease or other autoimmune disorders, adUTPase inhibitor can be used in combination with an antifolate orfluoropyrimidine or other thymidylate synthase and dihydrofolatereductase inhibitors; parasitic, viral or bacterial infections can betreated similarly employing a combination therapy including a dUTPaseinhibitor. Non-limiting examples of cancer include, colon cancer,colorectal cancer, gastric cancer, head and neck cancer, breast cancer,ovarian cancer, stomach cancer, lung cancer or a leukemia. The cancercan be metastatic or non-metastatic.

In one aspect, the compound or composition is administered as one ormore of: a first line therapy or alternativley, a second line therapy, athird line therapy, or a fourth or subsequent line therapy toadministration of a dUPTase-directed therapy. Non-limiting examples ofdUTPase-directed therapies include an antimetabolite or afluoropyrmidine therapy or a 5-FU based adjuvant therapy or anequivalent or each thereof, such as 5-FU, tegafur, gimeracil, oteracilpotassium, capcitabine, 5-fluoro-2′-deoxyuridine, methotrexate, orpemetrexed or an equivalent of each thereof.

Certain compounds provided herein demonstrated substantial, such as,5-100% dUTPase inhibitory effect, an ability to inhibit dUTPase underconditions described herein below, and/or known to the skilled artisan,compared, for example, to a positive control:

Kits

The compounds and compositions, as described herein, can be provided inkits. The kits can further contain additional dUTPase inhibitors andoptionally, instructions for use. In a further aspect, the kit containsreagents and instructions to perform the screen to identify patientsmore likely to respond to the therapy as described above.

Screening Assays

This invention also provides screening assays to identify potentialtherapeutic agents of known and new compounds and combinations. Forexample, one of skill in the art can also determine if the compound orcombination inhibits dUTPase in vitro by contacting the compound orcombination with purified or recombinant dUTPase in a cell free system.The purified or recombinant dUTPase and can be from any species, e.g.,simian, canine, bovine, ovine, rat, mouse or human. In one aspect, thedUTPase is DUT-N or DUT-M. Isolation, characterization and expression ofdUTPase isoforms are disclosed in U.S. Pat. No. 5,962,246 and known inthe art.

The contacting can be performed cell-free in vitro or ex vivo with acell or in a cell culture. When performed in vitro or ex vivo, thecompounds, compositions or agents can be directly added to the enzymesolution or added to the cell culture medium. When practiced in vitro orex vivo, the method can be used to screen for novel combinationtherapies, formulations or treatment regimens, prior to administrationto administration to an animal or a human patient. Methods to quantifyinhibition are known in the art, see, U.S. Patent Publ. Nos.2010/0075924 and 2011/0212467 and U.S. Pat. No. 7,601,702. For example,a fixed dose of a dUTPase directed therapy (e.g., 5-FU or Pemetrexed)can be added to the system and varying amounts of the compound can besubsequently added to system. Alternatively, a fixed dose of a compoundof this invention can be added to the system and varying amounts of thedUTPase directed therapy (e.g., 5-FU or Pemetrexed) compound can besubsequently added to system.

In another aspect, the assay requires contacting a first samplecomprising suitable cells or tissue (“control sample”) with an effectiveamount of a composition of this invention and optionally a dUTPaseinhibitor, and contacting a second sample of the suitable cells ortissue (“test sample”) with the agent to be assayed and optionally adUTPase inhibitor. In one aspect, the cell or tissue over expressdUTPase. The inhibition of growth of the first and second cell samplesare determined. If the inhibition of growth of the second sample issubstantially the same or greater than the first sample, then the agentis a potential drug for therapy. In one aspect, substantially the sameor greater inhibition of growth of the cells is a difference of lessthan about 1%, or alternatively less than about 5% or alternatively lessthan about 10%, or alternatively greater than about 10%, oralternatively greater than about 20%, or alternatively greater thanabout 50%, or alternatively greater than about 90%. The contacting canbe in vitro or in vivo. Means for determining the inhibition of growthof the cells are well known in the art.

In a further aspect, the test agent is contacted with a third sample ofcells or tissue comprising normal counterpart cells or tissue to thecontrol (or alternatively cells that do not over express dUTPase) andtest samples and selecting agents that treat the second sample of cellsor tissue but does not adversely effect the third sample. For thepurpose of the assays described herein, a suitable cell or tissue isdescribed herein such as cancer or other diseases as described herein.Examples of such include, but are not limited to cancer cell or tissueobtained by biopsy, blood, breast cells, colon cells.

Efficacy of the test composition is determined using methods known inthe art which include, but are not limited to cell viability assays orapoptosis evaluation.

In yet a further aspect, the assay requires at least two cell types, thefirst being a suitable control cell.

The assays also are useful to predict whether a subject will be suitablytreated by this disclosure by delivering a composition to a samplecontaining the cell to be treated and assaying for treatment which willvary with the pathology or for screening for new drugs and combinations.In one aspect, the cell or tissue is obtained from the subject orpatient by biopsy. Applicants provide kits for determining whether apathological cell or a patient will be suitably treated by this therapyby providing at least one composition of this invention and instructionsfor use.

The test cells can be grown in small multi-well plates and is used todetect the biological activity of test compounds. For the purposes ofthis invention, the successful candidate drug will block the growth orkill the pathogen but leave the control cell type unharmed.

The following examples are included to demonstrate some embodiments ofthe disclosure. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

EXAMPLE 1 Synthesis of Compounds Key Intermediate I(S)-1-azido-2-(3-(cyclopropylmethoxy)-4-fluorophenyl)butan-2-ol

Key intermediate I was prepared according to the literature data (J.Med. Chem. 2012, 55, 6427).

General Procedure A: Alkylation with LiHMDS

At −40° C., a solution of lithium bis(trimethylsilyl)amide 1 M intetrahydrofuran (38.9 mmol, 38.9 mL, 2.2 eq) was added dropwise to asolution of glutarimide (2.0 g, 17.7 mmol, 1.0 eq) in tetrahydrofuran(30 mL). The iodoalkane (53.1 mmol, 3.0 eq) was immediately added. After15 minutes at −40° C., the mixture was allowed to warm up and themixture was stirred at room temperature for 18 hours. The reaction wasquenched with a saturated solution of ammonium chloride (10 mL) and theaqueous phase was extracted with methylene chloride (3×20 mL). Thecombined organic phases were dried over magnesium sulfate, filtered andevaporated under reduced pressure. The residue was purified by flashchromatography using cyclohexane and ethyl acetate (100/0 to 0/100) toafford the expected compound.

General Procedure B: Alkylation with LDA

At 0° C., a solution of lithium diisopropylamide 2 M intetrahydrofuran/heptane/ethylbenzene (38.9 mmol, 19.5 mL, 2.2 eq) wasadded dropwise to a solution of glutarimide (2.0 g, 17.7 mmol, 1.0 eq)in tetrahydrofuran (30 mL). The iodoalkane (53.1 mmol, 3.0 eq) wasimmediately added. After 15 minutes at 0° C., the mixture was allowed towarm up and then stirred at room temperature for 18 hours. The reactionwas quenched with water (10 mL) and the aqueous phase was extracted withmethylene chloride (3×20 mL). The combined organic phases were driedover magnesium sulfate, filtered and evaporated under reduced pressure.The residue was purified by flash chromatography using cyclohexane andethyl acetate (100/0 to 0/100) to afford the expected compound.

General Procedure C: Reductive Amination

To a solution of the amino compound (HCl Salt) (1.0 eq) in methanol (10mL) was added a 7 N solution of ammonia in methanol (3.0 eq). Themixture was stirred at room temperature during 15 minutes and aceticacid was added until pH=5. The aldehyde (1.0 eq) and sodiumcyanoborohydride (3.0 eq) were added and the mixture was stirred at roomtemperature for 18 hours. The reaction mixture was carefully quenchedwith a saturated solution of sodium hydrogenocarbonate (10 mL). Theaqueous phase was extracted with ethyl acetate (3×15 mL). The combinedorganic phases were dried over magnesium sulfate, filtered andevaporated under reduced pressure. The residue was purified by flashchromatography using cyclohexane and ethyl acetate (100/0 to 0/100) toafford the expected compound.

General Procedure D: “Click Chemistry”

To a solution of the alkynyl compound (1.0 eq) and Key Intermediate I(1.0 eq) in dioxane (10 mL) degazed with argon was addedchloro(1,5-cyclooctadiene)(pentamethylcyclopentadienyl)ruthenium II (0.1eq). The reaction mixture was stirred at 80° C. for 3 hours. Aftercooling down, the reaction mixture was evaporated under vacuum and theresidue was absorbed on silica gel to be purified by flashchromatography using cyclohexane and ethyl acetate (100/0 to 0/100) toafford the expected compound.

EXAMPLE 1A2-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butyl)-morpholine-3,5-dione

Step 1:

2-hex-5-ynyl-morpholine-3,5-dione was prepared according to GeneralProcedure A using morpholine-3,5-dione (300 mg, 2.6 mmol) and6-iodo-1-hexyne (1.0 mL, 7.8 mmol). The expected compound was isolatedas colorless oil with 12% yield (65 mg).

Step 2:

The title compound was prepared according to General Procedure D, using2-hex-5-ynyl-morpholine-3,5-dione prepared in step 1 (65 mg, 0.3 mmol)and Key Intermediate I (93 mg, 0.3 mmol). The expected compound wasisolated as white solid with 53% yield (83 mg) after purification andlyophilization.

¹H NMR (CDCl₃): 8.02 (s, 1H), 7.39 (s, 1H), 6.94 (m, 2H), 6.78 (m, 1H),4.46 (m, 2H), 4.35 (d, J=14.0 Hz, 1H), 4.21 (d, J=14.0 Hz, 1H), 4.06 (m,1H), 3.79 (d, J=7.0 Hz, 2H), 2.32 (m, 2H), 2.00 (m, 2H), 1.84 (m, 2H),1.64-1.44 (m, 4H), 1.24 (m, 1H), 0.82 (t, J=7.2 Hz, 3H), 0.62 (m, 2H),0.34 (m, 2H)

EXAMPLE 1B4-(4-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-butyl)-piperazine-2,6-dione

Step 1:

4-hex-5-ynyl-piperazine-2,6-dione was prepared according to GeneralProcedure C using piperazine-2,6-dione hydrochloride (600 mg, 4.0 mmol)and hex-5-ynal (460 mg, 4.8 mmol, 1.2 eq) prepared from hex-5-yn-1-olaccording to the procedure described in the literature (US2011/306551).The 4-hex-5-ynyl-piperazine-2,6-dione was isolated with 50% yield (386mg).

Step 2:

The title compound was prepared according to General Procedure D, using4-hex-5-ynyl-piperazine-2,6-dione prepared in step 1 (150 mg, 0.8 mmol)and Key Intermediate I (216 mg, 0.8 mmol). The expected compound wasobtained as white powder after purification and lyophilization with 27%yield (100 mg).

¹H NMR (DMSO): 11.10 (s, 1H), 7.38 (s, 1H), 7.07 (dd, J=8.5 and 11.3 Hz,1H), 6.93 (dd, J=2.0 and 8.5 Hz, 1H), 6.83 (m, 1H), 5.29 (s, 1H), 4.41(s, 2H), 3.78 (d, J=7.0 Hz, 2H), 3.27 (s, 4H), 2.29 (m, 4H), 1.99 (m,1H), 1.77 (m, 1H), 1.58-1.38 (m, 4H), 1.17 (m, 1H), 0.69 (t, J=7.2 Hz,3H), 0.55 (m, 2H), 0.31 (m, 2H)

EXAMPLE 1C4-(3-{3-[(S)-2-(3-Cyclopropylmethoxy-4-fluoro-phenyl)-2-hydroxy-butyl]-3H-[1,2,3]triazol-4-yl}-propionyl)-piperazine-2,6-dione

Step 1:

To a solution of piperazine-2,6-dione hydrochloride (1.0 g, 6.6 mmol) inmethylene chloride (50 mL) was added pent-4-ynoic acid (782 mg, 8.0mmol, 1.2 eq), HOBt (1.2 g, 8.0 mmol, 1.2 eq), EDCI (1.5 g, 8.0 mmol,1.2 eq) and DIEA (3.4 mL, 19.9 mmol, 3.0 eq). The reaction mixture wasstirred at room temperature for 18 hours. Water (15 mL) was added andthe aqueous phase was extracted with methylene chloride (1×10 mL) andethyl acetate (2×10 mL). The combined organic phases are dried overmagnesium sulfate, filtered and evaporated under reduced pressure. Theresidue was purified by flash chromatography using cyclohexane and ethylacetate (100/0 to 0/100) to afford 4-pent-4-ynoyl-piperazine-2,6-dioneas white solid with 56% yield (725 mg).

Step 2:

The title compound was prepared according to General Procedure D, using4-pent-4-ynoyl-piperazine-2,6-dione prepared in step 1 (150 mg, 0.8mmol) and Key Intermediate I (216 mg, 0.8 mmol). The lyophilization ofthe purified residue afforded the expected compound as off-white solidwith 46% yield (169 mg).

¹H NMR (DMSO): 11.36 (s, 1H), 7.44 (s, 1H), 7.08 (dd, J=8.5 and 11.3 Hz,1H), 6.93 (dd, J=1.9 and 8.4 Hz, 1H), 6.86 (m, 1H), 5.30 (s, 1H), 4.51(d, J=14.2 Hz, 1H), 4.44 (d, J=14.2 Hz, 1H), 4.31 (m, 2H), 4.24 (m, 2H),3.81 (d, J=7.0 Hz, 2H), 2.65 (m, 4H), 1.99 (m, 1H), 1.74 (m, 1H), 1.17(m, 1H), 0.66 (t, J=7.2 Hz, 3H), 0.55 (m, 2H), 0.35 (m, 2H)

Other compounds of formula (I) and (II) are prepared in an analogousmanner. In some cases, protection of the “NH” group on thepiperidine-2,4-dione is required.

EXAMPLE 2 Preparation of Stereochemically Pure Compounds

The disclosed compounds can exist as two diastereomers. This exampledemonstrates a separation protocol. The stereochemical pure compoundsare prepared and then tested to determine if the biological activity isattributed to one or both stereoisomers.

Separation of the diastereomers is performed by preparative chiral highperformance liquid chromatography (HPLC) employing a 250×30 mm CHIRALPAKIA (5 μm) column, heptane/iso-propanol (70/30) with a flow-rate of 42.5mL/min and UV detection (λ=270 nm at 25° C.). Analytical chiral HPLC isperformed employing a 250×4.6 mm CHIRALPAK IA (5 μm) column,heptane/iso-propanol/diethylamine (70/30/0.1) with a flow rate of 1mL/min and UV detection (λ=230 nm at 25° C.).

EXAMPLE 3 Biological Methods

A. Drugs, Reagents and Cell Lines

Test compounds are suspended in DMSO at a concentration, e.g., of 100mmol/L, fluorodeoxyuridine (FUdR) that can be obtained from Sigma (StLouis, Mo.) and maintained in sterile double-distilled water at stockconcentrations of 50 mmol/L.

Recombinant human deoxyuridine nucleotidohydrolase (dUTPase) isexpressed and purified as described in Ladner R D, Carr S A, HuddlestonM J, McNulty D E, Caradonna S J. J Biol Chem. 1996 Mar. 29;271(13):7752-7. All drugs stocks are aliquoted and diluted asappropriate prior to use. The oligonucelotide primer, templates andfluorophore- and quencher-labeled detection probes are synthesized byIntegrated DNA Technologies (Coralville, Iowa), subjected topolyacrylamide gel electrophoresis purification and reconstituted inOmnipur sterile nuclease-free water (EMD Chemicals USA, Gibbstown N.J.)at a stock concentration of 100 μmol/L. The two non-emissive (dark)quenching molecules incorporated into the detection probes include theIowa black fluorescein quencher (IBFQ; absorption max 531 nm) and ZEN(non-abbreviation; absorption max 532 nm). The fluorescent labelutilized is 6-FAM (5″-carboxyfluorescein; excitation max.=494 nm,emission max.=520 nm). Probes are further diluted to a working stock of10 μmol/L and aliquoted to avoid repeated freeze/thaw cycles. AmpliTaqGold DNA Polymerase, GeneAmp 10×PCR Buffer 2, MgCl₂ and MicroAmp Optical96-well Reaction Plates are purchased from Applied Biosystems (Carlsbad,Calif.). dNTPs are purchased individually at stock concentrations of 100mmol/L from New England Biolabs at HPLC-certified >99% purity (Ipswich,Mass.).

B. Assay Components, Instrumentation and Real-time FluorescenceConditions

Reaction mixtures contained primer, probe and template at an equimolarfinal concentration of 0.4 μmol/L. Magnesium chloride (MgCl₂) isincluded at a final concentration of 2 mmol/L. Non-limiting dNTPs areincluded in the reaction mix in excess at a final concentration of 100μmol/L (dUTP/dTTP is excluded). AmpliTaq Gold DNA polymerase is added at0.875 U/reaction, 2.5 μl of 10×PCR buffer 2 added and nuclease-freeddH₂O added to a final reaction volume of 25 μl. For dUTP inhibitionanalysis, the volume of ddH₂O is further modified to accommodate anadditional 1 μl of dUTPase (10 ng/μl) and 1 μl of inhibitor or DMSOcontrol. Thermal profiling and fluorescence detection is performed usingthe ‘isothermal’ program on board an Applied Biosystems 7500 Real-TimePCR System. For analysis of dNTPs, the thermal profile consisted of an 8min 37° C. step followed by a 10 min 95° C. step to ‘hot-start’ the Taqpolymerase and a primer extension time of up to 30 min at 60° C.depending on the application. Raw fluorescence spectra for 6-FAM ismeasured using filter A at specified time intervals to follow assayprogression using Sequence Detection Software (SDS Version 1.4, AppliedBiosystems) and exported and analyzed in Microsoft Excel (Microsoft,Redmond Wash.) and Prism (GraphPad Software, La Jolla Calif.).Fluorescence values for blank reactions (limiting dNTP omitted) aresubtracted to give normalized fluorescence units (NFU) to account forbackground fluorescence.

C. MTS Growth Inhibition Assay

The Cell Titer AQueous MTS assay (Promega) is carried out according tothe manufacturers guidelines. IC_(50(72h)) values are calculated fromsigmoidal-dose response curves utilizing Prism (Graphpad, San Diego,Calif.). The combination effect is determined by the combination index(CI) method utilizing Calcusyn software (Biosoft, Ferguson, Mo.).Fraction affected (FA) is calculated from the percent growth inhibition:FA=(100−% growth inhibition)/100. CI values <1, synergism; 1-1.2,additive and >1.2, antagonism.

D. Colony Formation Assay

Colony forming assay showing the ability of colon (SW620, HCT116),non-small cell lung (A549, H460, H1299 and H358) and breast (MCF7)cancer cells to survive and proliferate following transient 24 hourexposure to test compounds, FUdR and combinations are determined.Specifically, cells are seeded at densities between 50 and 100cells/well in 24-well plates. Twenty-four hours later, cells are treatedwith increasing concentrations of a rtest compound, a fixed dose of FUdRand combinations of these. After 24 hours, drug is removed, cells arerinsed and allowed to outgrow for 10-14 days. At the conclusion of theoutgrowth, cells are fixed in 60% ice cold methanol and stained with0.1% crystal violet, scanned and counted. Data is presented aspercentage of untreated controls (mean±SD). Fraction affected andcombination indexes are calculated according to the method of Chou andTalalay where <1 is indicative of a synergistic drug interaction.

E. In Vivo Analysis

Xenograft experiments are conducted in male NU/NU nude mice (CharlesRiver, Wilmington, Mass.) that are 6-8 weeks old. Subcutaneous A549xenografts are established and allowed to grow until they reached ˜50mm³ (day 1). Animals are randomized to treatment groups: vehicle,pemetrexed 50 mg/kg, a test compound and combination of pemetrexed plusa test compound (n=5, group). Pemetrexed is administered at 50 mg/kg byintraperitoneal injection every two days. Test compound is administered,e.g., at 75 mg/kg by intraperitoneal injection every two days. Thecombination of pemetrexed and the test compound is administered byintraperitoneal injection, e.g., every two days. Two perpendiculardiameters of tumors are measured every 2 days with a digital caliper bythe same investigator. Tumor volume is calculated according to thefollowing formula: TV (mm³)=(length[mm]×(width [mm]²)/2. Mice areinspected everyday for overall health and bodyweight is measured every 2days as an index of toxicity. All animal protocols are approved by theUSC Institutional Animal Care and Use Committee (IACUC).

EXAMPLE 4 dUTPase Inhibition

Test compounds are screened in a fluorescence-based assay. The assayemploys a DNA polymerase-based approach utilizing an oligonucleotidetemplate with 3 distinct regions: a 3′ primer binding region, amid-template dUTP/thymidine triphosphate (TTP) detection region and a 5′6-Flavin adenine mononucleotide (FAM)-labeled probe binding region thatincorporates a black hole quenching moiety. During the reaction, theprobe and primer hybridize to the oligonucleotide template to form thetemplate:primer:probe complex. When Taq polymerase binds to the primerin the TPP complex and dUTP is present, successful extension of thenascent strand occurs and the inherent 5′ to 3′ exonuclease activity ofTaq polymerase cleaves and displaces the 6-FAM-labeled probe in a 5′ to3′ direction, releasing the 6-FAM fluorophore from its proximity to thethree quenchers. This displacement effectively disrupts the Försterresonance energy transfer (FRET) and the resulting fluorescence detectedupon excitation is directly proportional to the amount of the dUTPavailable in the assay for incorporation. Conversely, when the dUTP isunavailable, exhausted, or degraded by dUTPase and is no longeravailable for incorporation, Taq polymerase stalls and extension delayand/or chain termination of the nascent strand occurs. In this instance,probe hydrolysis/degradation does not occur and the probe remains darkas fluorescence remains quenched via FRET. Since fluorescence isdirectly proportional to the concentration of dUTP, the assay is easilymodified to measure dUTP and the effects of inhibitors on dUTPhydrolysis by the enzyme dUTPase. The template BHQ-DT6 (Black HoleQuencher—Detection Template 6) for detecting up to 60 pmols of dUTP isincluded for this application of the assay along with 50 pmols of dUTPand 5 ng of recombinant dUTPase. The reaction is incubated at 37° C. for8 mins and terminated by a 10 min incubation at 95° C. to simultaneouslyinactivate dUTPase and activate the hot-start Taq polymerase. Thefluorescence generated during the detection step is directlyproportional to the concentration of dUTP remaining after the 8 minincubation. The concentration of dUTP at reaction termination andtherefore inhibition of dUTPase in the presence and absence ofinhibitors and appropriate dimethyl sulfoxide (DMSO) controls can bedetermined.

EXAMPLE 5

Test compounds are evaluated for their antitumor activity in colorectalcancer cells using the MTS growth inhibition assay. HCT116 and SW620cells are exposed to increasing concentrations of each agent for 72hours and growth inhibition is directly compared to vehicle-treatedcontrols. The NSCLC cell lines A549 and H1299 are exposed to increasingconcentrations of each agent for 72 hours and growth inhibition isdirectly compared to vehicle-treated controls.

EXAMPLE 6 Growth Inhibition

MTS growth inhibition assays are performed to evaluate the effectivenessof the test compounds alone and in combination with the fluoropyrimidinethymidylate synthase (TS) inhibitor 5-fluorouracil (5-FU) at inhibitingthe growth of colorectal (HCT116 and SW620) cell line models. Increasingconcentrations of 5-FU between 0 and 100 μmol/L demonstrateddose-dependent increases in growth inhibition in both the colorectalcancer cell lines evaluated. Simultaneous treatment with increasingconcentrations of 5-FU and a test compound at fixed concentrations of 25μmol/L is determined.

EXAMPLE 7 Reducing Cancer Cell Viability

Colony forming assays are performed to evaluate the effectiveness oftest compounds alone and in combination with the fluoropyrimidinethymidylate synthase (TS) inhibitor fluorodcoxyuridinc (FUdR) atreducing cancer cell viability in colorectal (HCT116), breast (MCF-7)and non-small cell lung (H1299, A549, H358 and H460) cell line models.Increasing concentrations of FUdR between 0.5 and 2.5 μmon demonstrateddose-dependent decreases in colonies formed in all cell lines evaluated.In colorectal cancer cells, concentrations of test compounds ranginge.g., from 3.1 μmol/L to 50 μmol/L are combined with 0.5 μmol/L FUdR inHCT116 cells and 1 μmol/L FUdR in SW620 cells.

It should be understood that although the present invention has beenspecifically disclosed by certain aspects, embodiments, and optionalfeatures, modification, improvement and variation of such aspects,embodiments, and optional features can be resorted to by those skilledin the art, and that such modifications, improvements and variations areconsidered to be within the scope of this disclosure.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. In addition, where featuresor aspects of the invention are described in terms of Markush groups,those skilled in the art will recognize that the invention is alsothereby described in terms of any individual member or subgroup ofmembers of the Markush group.

The invention claimed is:
 1. A compound of formula (I):

or a tautomer thereof, including any stereoisomer, enantiomer ordiastereoisomer, or a pharmaceutically acceptable salt and/or a solvateof each thereof, of formula (I): or a tautomer thereof, or apharmaceutically acceptable salt of each thereof, wherein

is

Y¹⁰is O, S, SO, SO₂, NH or NR ¹⁵; R₁₁is hydrogen, halo, R¹² or —O—R¹²,wherein R¹² is C₁-C₆alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl optionallysubstituted with 1-3 hydroxy, fluoro, chloro, and amino substituent, orR₁₁ is C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl wherein each alkyl,alkenyl, and alkynyl is optionally substituted with 1-3 hydroxy, fluoro,chloro, and amino substituent; r is 1, 2, or 3; R¹⁵ is C₁-C₆ alkyloptionally substituted with 1-3 C₁-C₆ alkoxy, hydroxy, amino, and oxogroups; W is a bond or optionally substituted —CH₂—; X is a bond, O, S,NR¹⁹, optionally substituted C₁-C₆ alkylene, optionally substitutedC₂-C₆ alkenylene, or optionally substituted C₁-C₆ alkynylene group, adivalent optionally substituted C₆-C₁₀ aromatic hydrocarbon group, or adivalent optionally substituted saturated or unsaturated C₂-C₁₀heterocyclic or optionally substituted C₁-C₁₀ heteroaryl group; R¹⁹ ishydrogen, optionally substituted C₁-C₆ alkyl or optionally substitutedC₃-C₈ cycloalkyl; Y is an optionally substituted C₁-C₁₀ alkylene whichfurther optionally has a cycloalkylidene structure on one carbon atom,or is optionally substituted C₂-C₆ alkenylene, or optionally substitutedC₂-C₆ alkynylene group, or Y is -L¹⁰-B¹-L¹¹-; L¹⁰ and L¹¹ independentlyare optionally substituted C₁-C₆ alkylene, optionally substituted C₂-C₆alkenylene, or optionally substituted C₂-C₆ alkynylene group; B¹ is adivalent optionally substituted C₁-C₁₀ heteroaryl group; Z is—SO₂NR³¹R³², —NR³SO₂—R⁴, or R⁴ wherein R³¹ and R³² are the same ordifferent and each represents a hydrogen atom, optionally substitutedC₁-C₆ alkyl group optionally substituted with an aryl group; R³ ishydrogen or optionally substituted C₁ -C₆ alkyl; and R⁴ is optionallysubstituted C₆-C₁₀ aryl, an optionally substituted C₂ -C₁₀ heterocyclicgroup, or an optionally substituted C₁-C₁₀ heteroaryl group; providedthat if W is a bond, X cannot be a bond.
 2. The compound of claim 1,wherein each R¹⁹ independently is hydrogen or methyl.
 3. The compound ofclaim 1, wherein the uracil isostere A is:


4. The compound of claim 1, wherein the uracil isostere A is:


5. The compound of claim 1 of formula:

wherein A is:

R₁₁ is hydrogen, halo, R¹² or —O—R¹², wherein R¹² is C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl optionally substituted with 1-3 hydroxy,fluoro, chloro, and amino substituent, or R¹¹ is C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl wherein each alkyl, alkenyl, and alkynyl isoptionally substituted with 1-3 hydroxy, fluoro, chloro, and aminosubstituent; r is 1, 2, or 3, Y¹⁰ is O, S, SO, SO₂, NH or NR¹⁵, or X¹⁰is joined at Y¹⁰ with Y¹⁰ being a nitrogen atom; R¹⁵ is C₁-C₆ alkyloptionally substituted with 1-3 C₁-C₆ alkoxy, hydroxy, amino, and oxogroups; X¹⁰ is NH, O, or CH₂; and u is 0, 1, 2, 3, or
 4. 6. The compoundof claim 5, wherein A is:


7. The compound of claim 5, wherein A is: